barcoding t cell calcium response diversity with methods for automated and accurate analysis of cell...

7/17/2019 Barcoding T Cell Calcium Response Diversity With Methods for Automated and Accurate Analysis of Cell Signals

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Barcoding T Cell Calcium Response Diversity with Methods for Automatedand Accurate Analysis of Cell Signals (MAAACS)

Audrey Salles,#1,2,3,¤a Cyrille Billaudeau,#1,2,3,¤b Arnauld Sergé,1,2,3,*¤c Anne-Marie Bernard,1,2,3 Marie-Claire Phélip!,1,2,3 "iclas Ber!au,$,%

Ma!hieu &alle!,1,2,3 Pierre 'ren!,1,2,3 (idier Margue!,1,2,3 )ai-a )e,1,2,3and +annic )an1,2,3,*'régire Al!an-Bnne!, .di!r Au!hr in/ra!in 0 Ar!icle n!es 0 Cpyrigh! and icense in/ra!in 0his ar!icle has been ci!ed by !her ar!icles in PMC

AstractWe introduce a series of experimental procedures enabling sensitive calcium monitoring

in T cell populations by confocal video-microscopy. Tracking and post-acquisition

analysis was performed using Methods for Automated and Accurate Analysis of ell

!ignals "MAAA!#$ a fully customi%ed program that associates a high throughput

tracking algorithm$ an intuitive reconnection routine and a statistical platform to provide$

at a glance$ the calcium barcode of a population of individual T-cells. ombined with a

sensitive calcium probe$ this method allowed us to unravel the heterogeneity in shape and

intensity of the calcium response in T cell populations and especially in naive T cells$

which display intracellular calcium oscillations upon stimulation by antigen presenting

cells.

Author SummaryThe adaptive immune response to pathogen invasion requires the stimulation of

lymphocytes by antigen-presenting cells. We hypothesi%ed that investigating the

dynamics of the T lymphocyte activation by monitoring intracellular calcium fluctuations

might help explain the high specificity and selectivity of this phenomenon. &owever$ the

quantitative and exhaustive analysis of calcium fluctuations by video microscopy in thecontext of cell-to-cell contact is a tough challenge. To tackle this$ we developed a

complete solution named MAAA! "Methods for Automated and Accurate Analysis of

ell !ignals#$ in order to automate the detection$ cell tracking$ raw data ordering and

analysis of calcium signals. 'ur algorithm revealed that$ when in contact with antigen-

presenting cells$ T lymphocytes generate oscillating calcium signals and not a massive

and sustained calcium response as was originally thought. We anticipate our approach

providing many more new insights into the molecular mechanisms triggering adaptive

immunity.

!ntroductionalcium ion acts as a universal second messenger in response to most cellular stimuli ()*.

The pattern of the calcium response is biphasic$ and primarily results from the productionof inositol-+ phosphate ",+# which triggers the release of calcium from the endoplasmic

reticulum "/ store release# into the cytoplasm. This decrease is sensed by the stromal

interaction molecules "!T,M# that secondarily trigger the capacitative entry of

extracellular calcium via the calcium release activated channels "/A# of the '/A,

family (0* 1 (2*. Measuring the intracellular concentration of calcium is therefore of

primary interest when analy%ing transduction processes in living cells. urrently$ this is

achieved by methods which combine flow cytometry with intracellular diffusive

fluorescent calcium-sensitive dyes in immunological relevant cells such as macrophages$

34 cells$ T or 5 cells. As an example$ the calcium response is routinely monitored in T

cells (6* 1 ()6* as a functional read-out of the outside-in signal transduction subsequent to

T-cell receptor "T/# engagement by ma7or histocompatibility complex "M&#

molecules with agonist peptide. &owever$ when naive T cells encounter antigen-

http://www.ncbi.nlm.nih.gov/pubmed/?term=Salles%20A%5Bauth%5D


http://www.ncbi.nlm.nih.gov/pubmed/?term=Billaudeau%20C%5Bauth%5D


http://www.ncbi.nlm.nih.gov/pubmed/?term=Serg%26%23x000e9%3B%20A%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bernard%20AM%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ph%26%23x000e9%3Blipot%20MC%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bertaux%20N%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Fallet%20M%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Grenot%20P%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Marguet%20D%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=He%20HT%5Bauth%5D


http://www.ncbi.nlm.nih.gov/pubmed/?term=Hamon%20Y%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784497/citedby/


http://www.ncbi.nlm.nih.gov/pubmed/?term=Serg%26%23x000e9%3B%20A%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bernard%20AM%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ph%26%23x000e9%3Blipot%20MC%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bertaux%20N%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Fallet%20M%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Grenot%20P%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Marguet%20D%5Bauth%5D


http://www.ncbi.nlm.nih.gov/pubmed/?term=Hamon%20Y%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784497/citedby/




presenting cells "A# and more generally when signaling is induced by intimate

signaling-to-target cell-cell contact$ flow cytometry approaches cannot fully recapitulate

the physiological conditions of stimulation. ,n addition$ recent works have demonstrated

that T/ triggering by the M& molecules follows unusual physico-kinetic parameters

of serial engagement-disengagement ()8*$ ()9*$ which could be the molecular basis for

the broad selectivity$ high specificity$ extreme sensitivity ():* and the capacity to induce a

rapid intracellular response that characteri%e T/ triggering ();*. While soluble anti T/

or anti <+ antibodies (0=*$ antibody coated beads (0)*$ (00*$ and phorbol myristate

acetate>ionomycin (0+* can all induce a productive calcium signal in T cells that

ultimately leads to their activation$ proliferation and cytokine production$ the calcium

elevation triggered by these strong irreversible stimuli is usually sustained. ,t may not

therefore be representative of the response to physiological stimulations$ which is more

likely to consist in calcium spikes and oscillations (;*$ (02* 1 (08*. ,n order to capture the

true calcium responses triggered during cell-cell contacts such as those occurring during

T-cell and A stimulation$ video-imaging is compulsory in that it provides informative

parameters on individual cell behavior "i.e. displacements$ shape and intensity

fluctuation# (09*.

'btaining such imaging data requires a complex custom-built experimental set-up usually

dedicated to the detection of ?@-excitable calcium probes and to the maintenance of

physiological parameters for long-term recordings (;*$ (06*$ (0:*. ,n any case cell

tracking is mandatory and is often performed by manual approaches (0:* 1 (+=* however$

in addition to being time-consuming$ manual analysis is prone to systematic errors due to

sub7ective choice. !uch pre-selection is an unavoidable step in any manual analysis.

Automating the simultaneous tracking of hundreds of cells over hundreds of time frames

would overcome these issues. 3evertheless$ simultaneously tracking moving cells at high

density represents a considerable challenge$ particularly considering the need to correctly

resolve interlaced tracks of stretching cells while providing valuable statistical confidence

and robustness. While many software packages do incorporate a cell tracking module or plugins$ the normali%ation of the calcium signal for each cell as well as the classification

of calcium responses and any quantification generally have to be performed manually

involving tedious excel datasheets (+)*.

&ere we have developed a complete approach named Methods for Automated and

Accurate Analysis of ell !ignals "MAAA!# which enables the simultaneous tracking

of a population of individual moving cells "multiple target tracking$ MTT# (+0* and the

automatic extraction of robust statistics on pertinent observables. The MAAA! program

has been conditioned to synchroni%e$ normali%e and assemble the recorded cell traces to

provide an at a glance calcium barcoding of a heterogeneous cell population and facilitate

the a posteriori data mining and interpretation. We used MAAA! to examine the

calcium responses induced in T cells upon interaction with As and with it were able toreveal the oscillatory calcium responses in mouse naive <2B T cells upon antigen

recognition.

' !ResultsDevelopment of a protocol enaling the sensitive detection of intracellularcalcium fluctuations in TCR mediated T"cell stimulation via its naturalligand

Aiming to establish an easy$ robust$ sensitive and reliable way of evaluating calcium

fluctuations in T cells$ we assessed many visible calcium indicators such as Cluo-2 AM$

Cluo-+ AM$ and Cluo-:. All displayed short term leakiness of the loading without



membrane extruder blockers (++* "such as probenecid# (+2* and subsequent intracellular

compartmentali%ation$ incompatible with unbiased calcium measurements (+6*. ,n

contrast$ T-cell hybridomas "+A;# loaded with the calcium indicator 5< 5D appeared to

overcome most of these problems (+8*. ompared to standard loading conditions$ this

procedure provided a stable loading of the fluorescent indicator "emission spectrum fully

stackable with Cluo-2 AM Figure 1A#$ without affecting cell viability "Figure S1A#. We

also documented that 5< 5D fluorescence was photostable upon repetitive confocal

illumination for += min$ unlike Cura-red the fluorescence of which rapidly decreased

"Figure S1B#. This precluded us from performing 5< 5D>Cura red ratiometric cytosolic

calcium measurements (+9*$ (+:*. ,n addition$ we investigated whether cytosolic calcium

gradients could be visuali%ed under such experimental conditions since few discrete

hotspots were detectable among the homogenous fluorescence. 5< 5D and mitotracker

red loaded cells were imaged to decipher whether mitochondria would accumulate

calcium indicator. ,n +A; T cells the two signals were not mutually exclusive "Figure

S1.C,D# unlike in Eurkat cells or primary human T cells (+6*$ (+;*. art of the signals was

correlated under stimulation$ most presumably caused by C/T between the two dyes

"Figure S1.C, D#. We detected no significant sequestration of the 5< 5D calcium dye$unlike Cura-0 in Eurkat cells although this phenomenon had a limited impact on whole

cell calcium measurement (+6*.

Cigure )

BD PBX, a highly sensitive calcium probe allows ratiometric analysis an

antiboy labeling o! primary cells. "A# Comparison o! e$citation anemission spectra o! Fluo%&%A' an the calcium inicator inclue in the

PBX calcium assay (it, BD PBX.

These inconsistencies with previous reports (+2* motivated us to consider 5< 5D as a

close relative of Cluo-2 AM but harboring subtle differences$ that required the dedicated

loading buffer to avoid the dye leakage displayed by Cluo-2 AM "Figure S1A#. <ue to

the lack of information about the 5< 5D from the manufacturer$ we thus determined the

in vitro 4d of the 5< 5D as described previously (2=* "Figure 1B#$ which gave a

consistent value for 4d of +)0 nMF++$ comparable to the 4d for Cluo-2 AM of +09F2;

nM and that in the literature "4dG+26 nM# (2)* "see Materials and Methods#. The great

similarity between 5< 5D and Cluo-2 AM led us to assume the in situ 4d of 5< 5D

to equal the reported in situ 4d value ") HM# of Cluo-2 AM (+2*$ deemed acceptable

when accurate determination by electrophysiology is either not feasible or not available

(0:*. 5ased on this 4d value$ we estimated that the intracellular calcium concentration in

resting +A; T cell hybridomas would be around ;= nM and consistent with previously

published values for these hybridomas (20* as well as leukemic cell lines Eurkat (+;*$

mouse thymocytes (2+* and human peripheral blood lymphocytes (+6*.

We used flow cytometry to compare the detection sensitivity of 5< 5D with the

ratiometric ,ndo-) AM "routinely used in calcium assays$ ?@ excitable#. ,n terms of

response pattern$ T cell hybridomas loaded with 5< 5D do not strictly speaking respond

the same as those loaded with ,ndo-) AM$ as the non-ratiometric calcium indicator 5<

5D does not allow the evaluation of intracellular free calcium concentration "Figure

1C#. We therefore sought to estimate the intracellular calcium concentrations in 5< 5D

loaded T cell hybridomas. ?pon various concentrations of ionomycin$ we compared

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784497/figure/pcbi-1003245-g001/













intracellular calcium elevation in 5< 5D versus ,ndo-) AM loaded +A; T cell

hybridomas. As previously mentioned$ the kinetics were not fully stackable and the

calculated intracellular calcium concentration differed between the two methods due to

4d discrepancy and loss of linearity in the relationship linking calcium concentrations

and high fluorescence amplitudes (2=*. ,ndeed at such high fluorescence values under

strong ionomycin concentrations$ the calcium concentration was overestimated and is the

reason for us reporting fluorescence amplitude instead of erroneous calcium

concentrations throughout this manuscript. 3evertheless to our surprise$ lower

concentrations of ionomycin rapidly abrogated fluorescence elevations of indo-) AM

whereas 5< 5D fluorescence elevation remained detectable even at subnanomolar

ionomycin concentrations. This indicated that 5< 5D is sensitive to low intracellular

calcium elevation. Thapsigargin "Figure 1D# or the cross-linking of the T/><+

complex by anti-<+I "0)) biotin>streptavidin# induced in cells loaded with 5< 5D

responses similar to those induced by ionomycin. This method was extendable to naive

primary <2B T cells labeled or not with an anti <2 monoclonal antibody "Figure 1)#.

Automatic trac#ing of high density moving cells y MAAACSWhile surface receptor crosslinking with antibodies is a convenient way of stimulating

calcium responses in T cells$ it cannot physiologically reproduce the dynamics of

T/>M&-antigen interactions in the context of T-cell>A contacts. 'ur goal was to

decipher calcium signals arising from cellular contacts and requiring imaging approaches.

We chose to perform all recordings on a conventional argon laser equipped-confocal

scanning microscope$ widely found in laboratories. onsidering the lack of available

methods (22* able to combine automatic tracking of cells and calcium signal processing$

we set up our own procedure to automatically track moving cells at high densities with a

minimum of input parameters. ,t is a customi%ed version of our previously developed

MTT algorithm (+0*$ originally dedicated to tracking single fluorophores coupled to

plasma membrane molecules at high density. We built a plugin that converts cellularimages into cell position images that are comparable with the single molecule images

supported by MTT "Figure *A + Figure S*#. /aw fluorescence images were first passed

through a median filter to eliminate the electronic noise emanating from detection and an

appropriate mask$ consisting of a disk of adequate radius "see Materials and Methods for

details on mask si%e#$ was applied to identify cells as single ob7ects. ach cell was thus

defined by the xy coordinates of its centroid which then served to reconstruct the cellJs

tra7ectory over the stack of images "Figure *A + ieos S1, S*, S-, S&, S, S/, S0#.

3oteworthy$ more complex cellular shapes could be handled if using other appropriate

detection schemes. 3ext$ we generated synthetic images containing Kaussian peaks at the

corresponding positions$ with a radius optimi%ed for the tracking performed by MTT and

an intensity equal to the integrated cell signal$ itself proportional to the intracellular

calcium concentration. The resulting sequence of single molecule like images could then be treated by MTT "ieos S, S/#. /eplacing each cell by a Kaussian peak of smaller

si%e prevented the occurrence of two targets crossing over each other$ initially a ma7or

concern for MTT$ thus rendering the reconnection of traces during the MTT procedure far

more efficient. 'verlapping was then handled at the detection stage$ where crossing cells

presenting as a Lpeanut shape were detected as two targets. &owever$ strongly

overlapping cells resulting in more of a spherical shape were detected as a single target

and thus required %-stack acquisitions and appropriate analysis to recogni%e the

occurrence of such crossing tra7ectories.











Calcium signal processing. "A# Automatic

trac(ing o! high ensity moving cells by

'AAACS.

Automatic signal analysis y MAAACS

MAAA! generates traces which are defined by the position of the detected cells overtime and intrinsically calculates their instantaneous velocity and their fluorescence

intensity. Kiven that the basal level varies from cell to cell due to differences in

intracellular calcium concentrations$ or due to heterogeneous efficiency in calcium

indicator loading$ we needed to accurately set a baseline of fluorescence that we defined

as the median of fluorescence calculated until the maximum fluorescence value had been

reached for each cell "Figure *B#. To establish the best mode of normali%ation$ we

analy%ed for each cell in non-stimulating conditions how the mean signal amplitude was

correlated with signal fluctuations. We found that this relationship was proportional$ thus

implying that the normali%ation can be performed by division. alcium responses are

highly diverse$ both in terms of magnitude and oscillation "Figure S-#. The amplitude of

calcium mobili%ation varies according to the type of stimulus and the addition of

inhibitors$ but also within a cell population for a given stimulus>treatment. Moreover$ the

shape of these signals$ their maintenance and their oscillations are also varied. We

therefore defined analytical parameters to describe and characteri%e these response

diversities "Figure *C#. Cor each cell$ the response magnitude is described as the

fluorescence amplitude (FA) of calcium mobili%ation$ corresponding to the time-average

of normali%ed intensities on the whole trace. The temporal fluctuations are deciphered by

analy%ing the persistence and the oscillations of the calcium signals. We defined as the

responsefraction (RF) the ratio of two phasesN the time when the normali%ed intensity is

greater than the threshold over the total time during which the intensity is detected. We

also calculated the number of bursts/min "BP'# defining the number of peaks detected

above the threshold divided by the duration of the detected trace "able S1#. ,n order to

provide a global$ comprehensive view of the calcium response in a substantial number ofcells for any given condition$ we color coded the normali%ed calcium intensities with a

gradient of blue and orange for values below or above the threshold "see below#$

respectively. The resulting values$ for each cell at each time-point$ could then be pooled

to generate a heat map$ the dimensions of which hence corresponded to the cell number

and time "Figure *C le!t panel#. 3on-relevant pixels$ either before or after detection of a

given cell in the time-lapse movie$ were left in black. This representation simultaneously

depicts the global tendency$ together with the intra-population variability of response

(26*. ollectively$ all calcium signal parameters are summari%ed on scatter dot plots

where responding cells are represented by a single dot "Figure *C right panel#. 5y

integrating these different parameters$ the heterogeneous behavior of activated cells

"maintained$ oscillatory and unique# can be determined and inactive cells identified$ the

proportions of which are then represented in a pie chart diagram "see Materials and













Methods for details on the classification#.

MAAACS trac#ing performance versus e$haustive manual trac#ing

An endemic problem in automatic tracking approaches is reaching a level of

completeness that manual tracking only can guarantee. We analy%ed several videos in parallel by the automatic and by manual methods and determined the percentage of cells

tracked by MAAA! compared to that by manual tracking in the observation field

"detection percentage#. +A; T cell hybridomas or naive <2B T-cells loaded with 5<

5D were seeded onto a monolayer of '!-9 cells stably expressing the molecules of the

ma7or histocompatibility complex (28*. We chose experimental situations with high cell

densities on a rough and irregular surface. We only considered tracked cells detected by

MAAA! over more than 6 images. The superimposition of the tra7ectories obtained

manually or with MAAA! "Figure -A# illustrated the efficiency of our algorithm$ the

detection percentage of which was greater than ;8O "3G)06#. !urprisingly$ more traces

were generated by MAAA! than obtained manually. onsequently$ the MAAA! cell

traces were fragmented into several parts as shown in Figure -B illustrated by the

number of fragments needed to reconstitute the full length tra7ectories ").+ for primary T

cell and ).; for hybridomas$ Figure -B#. The lower efficiency for the latter can be

explained by significant cellular shape changes over time preventing their detection by

the circular mask and thus their reconnection. To fully document the cell response over

time and to improve the quantitative analysis of cell signaling$ we implemented a

program allowing the reconnection of fragments of tra7ectories "Figure -C#. Cirst$ the

method selected among the set of tra7ectories terminating before the end of the

acquisition "plotted in gray in Figure -C# to be reconnected to traces starting after the

breaking off "plotted in red and blue in Figure -C#. Then the algorithm classified the

candidates for reconnection by minimi%ing the interval between the stop and start times

"Pt#$ the distance between the final and initial positions "Pr# and the difference of the

mean fluorescence amplitude of the fragments "P,#. The user is free to decide whether thetra7ectories should be reconnected by consulting the original video. ,n this way$ the

tracked time percentage was clearly improved ";6O for primary T cells and :+O for

hybridomas$ Figure -D#.

'AAACS trac(ing per!ormance versus e$haustive manual trac(ing. "A# 2verlay o!

cell tra3ectories analy4e manually or with 'AAACS.Threshold of specificactivation

Within the course of this study$ we noticed that without specific stimulation$ cytoplasmic

calcium concentrations displayed spontaneous oscillations$ the amplitude of which was

almost negligible for T cell hybridomas but not for primary naive T cells ()+*. To account

for this$ we performed a median filtering with a sliding window of 9 frame-si%e on eachfluorescence amplitude to remove aspecific oscillations in the absence of any stimulus. ,t

















was then important to carefully define the threshold of peptide-specific activation. As

mentioned by many authors (08*$ (20*$ (29* and in our observations "Figure -#$ calcium

signaling exhibits high diversity even within the same cell line and depends on the

applied perturbations "stimulation$ drug treatment or mutation#. ,t should be noted that

defining any biological threshold for activation could be misleading since it is highly

dependent upon the experimental conditions. Moreover$ the definition of a criterion for

specific activation should respect the response heterogeneity without favoring a subset of

responding cells. Accordingly$ we set up a detector which compares the fluorescence

amplitude to a threshold of activation in order to identify activated cells in our

experiments "Figure S&#. ,f the fluorescence amplitude is greater than the threshold$ then

the cell is declared as activated. To determine this threshold$ we investigated the

statistical properties of the fluorescence amplitude of cells in the absence of any

stimulation compared to that in cells that have been activated as a result of stimulation.

Cor a given probability of false alarm "CA#$ an activation threshold could be deduced

from the cell responses in the absence of any stimulation. The probability of detection

"<# could then be calculated as the percentage of activated cells revealed by the detector.

We then aimed to identify threshold values that minimi%ed false detections "low CA#without decreasing the identification of activating cell "high <#. A robust method to

ob7ectively determine the activation threshold is to perform a receiver operating

characteristic analysis "/' curves in Figure S&A, B# to explore the values of activation

threshold that maximi%e the overall score < x ")-CA#. All activation thresholds are

reported in Figure S&C. !urprisingly$ the activation threshold was quite stable whatever

the cell type "hybridoma or naive T cells# or stimulation process "antibody or A#. Cor

T-cell hybridomas$ this method exhibited very high < "Q=.;;# and low CA "R=.=0#. <

was also high in primary T cells "Q=.;:# and the CA was reasonable "R=.=:# though

slightly higher due to a higher diversity in the cell signaling.

%valuation of the calcium response of a heterogeneous population of

individual T cell hyridomas

To test our MAAA! algorithm$ we analy%ed a population of T hybridomas +A; loaded

with 5< 5D and seeded at the bottom of a well of a Sab-Tek before their stimulation

with thapsigargin after ) minute. We generated a sequence with a frequency of ) confocal

image every 9 seconds for += minutes. Cluorescence intensity was not affected by

repetitive illuminations as previously mentioned "Figure S1B#. 3o a priori assumption

was made and raw recordings were sub7ected to MAAA!. To compare the calcium

signals$ we normali%ed the fluorescence intensities as a fold of the basal fluorescence. A

mean curve of variations in fluorescence over time was obtained "Figure &C# and

compared to flow cytometry measurements "Figure &B#. Cluorescence rose immediately

after thapsigargin addition$ reaching a plateau + minutes after induction (2:* before

slowly decreasing "Figure &C#. This response was fully stackable with the kineticsmonitored by flow cytometry "Figure &B#. We noted that the response>baseline ratio was

higher according to imaging recordings$ presumably due to a better sensitivity of

detection on confocal photomultipliers. ,n this case the benefit of MAAA! is limited

since all individual cells responded homogenously by a strong$ sustained non-oscillating

response "fluorescence amplitude$ CAG9.;+F=.6= response fraction$ /CG=.;)F=.=0

bursts per minute$ 5MG =.=2F=.==)#. ,n the presence of the /A channel blocker 0-

aminoethoxydiphenyl borate "0-A5# (2;*$ thapsigargin induced a weak elevation of

fluorescence that was similar to a0B>Mg0B deficient incubation conditions (2:* "Figure

&A# and consistent between the two methods "Figure &B, C#. The analysis of the global

tendency clearly masked the singularity of each individual cell. Through MAAA!

analysis$ while 6=O of the cells displayed a unique rise of fluorescence as the average

tendency would have suggested$ the other half was equally composed of cells displaying



















sustained or oscillatory behavior. This implies that within a cell population and for any

given stimulus$ the observed differences in response are not limited to intensity dispersion

but also to the mode of response. We sought to document this point using different

experimental stimuli each producing their own calcium response in terms of shape$

intensity and heterogeneity "Figure # (6=*. ,ndeed$ when the cells were seeded onto an

activating surface "anti T/ antibody coated pits#$ while most responses were sustained

"CAG2.2F=.2+$ /CG=.80F=.=+$ 5MG=.=;F=.=)#$ heterogeneous responses were also

observed. These heterogeneities were even more obvious when the +A; cells were

stimulated by ,-A4-&S expressing '!-9 As. The asynchronous landing of the T

cells and the heterogeneous M& ,, agonist peptide expression levels are parameters that

affect the calcium response in addition to irregular crawling and scanning activities of the

T cells on an A monolayer. ,ndeed$ during the first += minutes$ 6=O of the cells

displayed a specific calcium rise ()6*. Most of these exhibited a maintained fluorescence

amplification but which was weaker in term of intensity and response fraction as

compared to that in response to the stimulating antibody "Figure C#$ supporting the

notion that abundant$ immobile$ highly affine ligands are not strictly recapitulating the

stimulation by the natural membrane ligand of the T/. !upporting this view$ weanaly%ed the cell motility by MAAA!$ as an integral parameter of T cell activation (0;*$

(20*$ (6)*. MAAA! analysis of cell velocity showed that inducers of strong and

sustained calcium responses "thapsigargin or anti T/ coated slides and to a lesser extent

anti <26 unstimulating surfaces# negatively impacted the motility of cells ()0*$ since

instant speed measurements did not exceed 0 Hm>min in the few minutes after landing on

the slide$ indicating that the cells were almost immobile. ,n contrast$ T-cells migrating on

'!-9 ,-A4-&S displayed higher velocities "unactivatedN 6.)F0.2 Hm>min activatedN

+.9F).0 Hm>min# with a high mobile fraction "unactivatedN =.2:F=.)9 activatedN

=.68F=.)8#. These velocities are fully consistent with 0-photon-microscopy measurements

"Figure S#$ where migratory T cells in lymph nodes$ or thymus slices display mean

velocities around 2 Hm>min (;*$ (0:*$ (60* 1 (62* or stimulating lipid bilayers (66*.

*%APB inuce inhibition o! C5AC channel activity prior to thapsigargin

stimulation evaluate using !low cytometry as compare to con!ocal imaging

analysis using 'AAACS. "A# 6ntracellular calcium mobili4ation measure in 7BSS

without calcium an magnesium ...







)valuation o! calcium response o! a heterogeneous population o! iniviual cell

hybriomas upon i!!erent e$perimental stimuli.Although experimental$ '!-9 ,-A4-

&S have been shown to efficiently simulate physiological situations of T cell activation

by inducing productive calcium signals$ in a context of immunological kinapses (68*$ (69*rather than stable immunological synapses leading to specific cytokine secretion such as

,nterleukin-0 "data not shown#. Additionally$ as previously demonstrated$ a clear

correlation exists between activation and motility since unactivated T cells appear to

move faster than activated ones within the same population (6:* and T cell hybridoma

mobility appears to decrease rapidly after calcium rise and rounding of the cells ()=*$ ()0*

"Figure S/A#. MAAA! was conditioned to automatically analy%e the velocity and

shape of the cells in addition to fluorescence signals$ although no link between these

parameters was found that was as tight as previous reports in cell systems expressing co-

stimulatory or specific adhesion molecules.

,n the presence of 0-A5$ the fluorescence amplitudes upon either anti T/ "CAG

+.08F=.02# or '!-9 ,-A4-&S stimulation "CAG).68F=.=8# were strongly reducedcompared to /A active control conditions. &owever$ although the response fraction

and the number of bursts per minute were left unaffected "/CG=.68F=.=+$ 5MG

=.=;F=.=0# "Figure C# upon anti-T/ T cell activation$ short and low calcium

oscillations (08* were predominant in most T-cells "/CG=.08F=.=)$ 5MG=.)2F=.=)#

"Figure C an Figure S-B# seeded onto '!-9 ,-A4-&S.

,n this case$ the lack of co-stimulatory or specific adhesion molecules that usually sustain

T cell>A interactions and signaling (6;* suggested that following T/ engagement by

M&-peptide$ signaling events would occur through waves such as displayed by the

calcium oscillations (8=*. We therefore titrated the T/-dependent calcium signaling in

the presence of 0-A5 in T-cells as a function of the amount of peptide loaded onto '!-

9 ,-A4. As shown in Figure /$ the peptide-specific$ 0-A5 sensitive calcium response

was dependent upon the amount of peptide presented by '!-9 ,-A4. ,n the absence of

peptide$ about 6O of the cells displayed a weak but significant calcium response above

threshold. The percentage of responding cells increased proportionally to the peptide

concentration to reach a plateau at a &S 2818) peptide concentration of 6= nM "Figure

/A#$ while being constantly oscillatory "Figure /)#. The fluorescence amplitude of the

responding cells was significantly higher than that observed in the absence of antigenic

peptide$ even at low antigen doses "down to =.6 nM#. "Figure /B# !urprisingly$ the

fluorescence amplitude$ response fraction$ and burst frequency were independent of the

antigen concentration "except at higher antigen concentrations# "Figure /B8D#. These

data show that$ at least in +A; T cell hybridomas$ the T/-mediated antigen-dependent

/-store-operated calcium response is digitally triggered irrespective of the antigenic peptide concentration. This is consistent with results from an earlier study (20* focusing

however on the global calcium responses in +A; T cells.





















)!!ect o! peptie concentration on the )5 release o! calcium.&aive CD' T cellsdisplay heterogeneous intracellular calcium flu$es

The antigen-dependent calcium response of mouse primary T cells has previously been

investigated in vivo by microscopy on explants or sections of lymphoid organs (;*$ (0:*$(6+* or ex vivo on artificial activating surfaces (66*. &owever$ most of these studies were

performed on lymphoblasts obtained by continuous activation in the presence of ,S-0 for

several hours and which therefore differ from naive T cells ():*$ (29*$ (8)*. <ata from

studies that have examined the calcium response of naive T cells suggest that the calcium

homeostasis of naive <2B T cells ex vivo is complex and at least in part antigen-

independent ()+*$ (80*. We evaluated these calcium responses of T-cells with MAAA!

"Figure 0#. 3aive +A; transgenic <2B T cells (8+*$ (82* seeded onto a surface coated

with anti T/ antibodies showed a strong increase in fluorescence "CAG2.8)F=.06# that

was maintained over time "/CG=.9)F=.=+$ 5MG=.)+F=.=)# similar to that observed

with +A; hybridomas "Figure 0A, B,

$C#.

C#. &owever$ we also observed that when seeded onto non-stimulating ,-A4 expressing

'!-9 cells$ around 0=O of naive T cells responded spontaneously with short weak

calcium pulses "CAG).0+F=.=+$ /CG=.)+F=.=) 5MG=.)2F=.=)# reminiscent of those

previously reported ()+* and (;*. eptide specific calcium signals in the presence of '!-

9 ,-A4-&S were mostly oscillatory in more than 8=O of the cells "CAG0.=0F=.=;$ /CG

=.+0F=.=0$ 5MG=.00F=.=)#$ in contrast to those observed in hybridomas " Figure 0B#

which were mainly sustained. Another fundamental difference with hybridomas is that we

found no clear correlation between calcium fluxes and cell velocity "Figure S/B#$ which

nevertheless was expected considering in vivo reports (86*. We then wondered whether

these calcium responses in <2B naive T cells were sensitive to 0-A5. /A channel

activity in T cells is characteri%ed upon stimulation by thapsigargin or soluble anti-<+antibody "0))#$ generating sustained calcium responses that are absent in patients

suffering from an inherited form of severe combined immune deficiency "!,<#

syndrome or upon 0-A5 treatment (+* ,ndeed$ upon 0-A5 treatment$ the thapsigargin-

induced calcium response was drastically reduced. quivalent kinetics "evaluated by flow

cytometry# was obtained with 0)) stimulation in the presence of 0-A5 or <TA

"Figure S0A8B#. 3o additive or competitive effect was detected under these

experimental conditions "Figure 0C#. ,n fact$ 0-A5-treated naive T cells seeded onto

anti T/ coated surfaces did show a calcium response "CAG0.==F=.)2$ /CG=.+0F=.=+$

5MG=.)+F=.=)# that was greatly reduced compared to the native conditions without

/A inhibitor "Figure 0A#. More unexpectedly$ 0-A5 treatment did not induce a

unique calcium peak suggesting that other calcium channels than !' mediate calcium

entry in naive mouse T cells$ since calcium oscillations are dependent upon calcium



















influx (88* 1 (8:*. !imilarly$ we analy%ed the calcium response to '!-9 ,-A4-&S in the

presence of 0-A5. There was a moderate but significant decrease in the amplitude of the

calcium response "CAG).88F=.=9$ /CG=.0;F=.=)$ 5MG=.)8F=.=)# compared to

conditions in absence of 0-A5$ together with a slight decrease of the oscillation

frequency "Figure 0A8C#. ,n addition 0-A5 on naive T-cells seeded onto '!-9 ,-A4

did not show any significant impact on calcium fluxes "CAG).0:F=.=6$ /CG=.)+F=.=0$

5MG=.)6F=.=0#$ remaining lower to the calcium response in presence of antigenic

peptides. Altogether upon blockade of the /A channel activity by 0-A5$ we evidence

that the !' dependent calcium entry plays a limited role in mouse naive T-cells upon

T/ triggering by-M&-peptide.

9aive CD&: cells isplay mainly intracellular calcium oscillations upon antigenic

challenge. "A# Barcoing o! stimulation.' !DiscussionThe first aim of this study was to significantly increase the sensitivity$ accuracy$

completeness and statistical reliability of video-microscopy approaches to record calcium

fluxes$ by combining a strong calcium probe with a robust algorithm for high density cell

tracking coupled to an automated interface for rigorous post-acquisition analysis. The

second ob7ective was to use this method to describe the characteristic parameters ofintracellular calcium fluxes within a population of T cells in response to different stimuli.

We highlighted the heterogeneous nature and dynamics of these fluxes after T/

engagement by its natural ligand in a cell>cell context$ which cannot be documented by

flow cytometry. The T/-M&-peptide is a paradigm for unconventional intercellular

receptor-ligand interaction (8;* based upon successive cycles of engagement>release ()8*$

()9*. &owever$ more functional data supporting this current view are needed$ taking care

to account for the free motility of cells prospecting for cognate antigens supported by

M& molecules in a 0< cell membrane environment. 'ur goal was to develop

experimental tools to contribute to the understanding of these mechanisms. The first

challenge was finding a bright and stable fluorescent calcium probe in the visible range of

the spectrum and that was easy to monitor both by cytometry and on conventionalconfocal microscopes.

T cells dedicated to calcium imaging are usually loaded with calcium indicators the

emitted fluorescence of which is ?@-shifted upon calcium elevation thus allowing

ratiometric measurements "such as Cura-0#. The sensitivity of these probes can however

be impacted by their intracellular compartmentali%ation "adsorption by proteins$

interaction with membranes or sequestration by organelles e.g. mitochondria#$ or their

extrusion by organic cation transporters. To overcome these technical issues$ loading can

be performed with diluents "pluronic acid# or transporter blockers "probenecid#$ although

these compounds can be noxious to T-cells and thereby affect their response (+:*. <espite

these potential drawbacks$ Cura-0 loaded T cells are routinely used for long experimental

procedure followed by transcriptomics without limitations by reduced cell viability (06*.





,n our study$ cells loaded with 5< 5D were used over extended periods of time without

any evidence of mortality$ compartmentali%ation$ or photobleaching which have been

reported to affect Cluo-2 AM use (+2*. Although they could be considered as anecdotic or

trivial$ such properties enable more reproducibility and the use of 5< 5D in a greater

number of experiments compared to other fluorescent visible probes. 'ur here proposed

MAAA! method incorporates our previously reported MTT algorithm dedicated to

single particle tracking (+0* and nanoscopy (9=*$ (9)* set up to enable the detection$

monitoring and reconnection of tra7ectories of moving T cells acquired by conventional

confocal microscopy. The ability to simultaneously track a great number of targets is in

itself a challenge but in particular encounters difficulties when tracks are interlaced or

crossing over. The performance of MTT was found to be slightly superior compared to

existing algorithms however the implementation of a program of assistance proposing

candidate traces to be reconnected to aborted traces was a ma7or breakthrough in terms of

improving the accuracy and completeness. ,n addition$ during the analysis process$

MAAA! enabled the re7ection if necessary of dead or dividing cells. onsequently$

while MAAA! is not yet a fully unsupervised method$ we speculate that +< time lapse

video-acquisition methods "on a spinning disk confocal microscope$ for example# wouldgreatly reduce the number of aborted traces due to focus loss that occurs on a 0<Btime

acquisition scheme such as that in this study "in particular for primary T cells#.

ompleteness is a ma7or issue in this kind of study$ since the baseline calculation could

be under-estimated or incorrect when the first time points after cell landing are missing.

&ere$ the automated normali%ation of calcium signals facilitates their comparison among

a population of cells. The MAAA! analysis makes simple the analysis and$ more

importantly$ the quantification of signaling. MAAA! deciphers a video sequence in

about )= minutes$ where manual tracking and analysis would take at least 0 hours

"depending on the duration of the time lapse and the number of cells#. @ideo microscopy

records the behavior of individual cells over time and not 7ust part of a population of

anonymous cells. This allowed us to demonstrate that calcium oscillations are highly

diverse among cells ()6* both in terms of intensity and frequency they are mostlytransient oscillations in primary T cells in contact with antigen loaded As. This

diversity in cell responses supports the notion that T-cell triggering is stochastically

linked to heterogeneity in the T cell population (6=*. This is conceivable for T-cell clones

due to genomic drift$ but may seem more surprising for primary <2B T cells. Siterature

reports that oscillatory calcium fluctuations are associated to effector function of T cells

and proliferation (0)* in contrast to memory T cells which display unique increases in

calcium (90*. ,n addition$ sustained calcium responses are observed mainly in apoptotic T

cells (9+*. Altogether$ our approach would be able to reveal in a seemingly homogenous

population$ T cell diversity in terms of function or fate$ based upon antigen dependent

calcium response mode. Another interesting finding is that /A channel dependent

activity does not support a sustained calcium response in naive T cells encountering

As$ and that the predominant calcium response modes in T cells are oscillations$ inagreement with literature (6*$ (;*$ (06*$ (92*$ at least in part sensitive to 0-A5 blockade.

This result supports recent works showing an intriguing role of voltage dependent a0B

channels "av).2# (88* 1 (8:* in the calcium influx into naive T cells. 'ur results also

suggest that membrane calcium channel openings are tightly correlated to /-calcium

waves upon T/ triggering (96*$ (98*$ and that sustained calcium fluxes such as those

triggered by stimulating antibodies and revealed by flow cytometry or video imaging are

not strictly physiologic$ at least not in naive T-cells. ,t could be valuable to consider our

results in the light of recent evidence suggesting a role for cytoplasmic calcium sustained

elevation in the orientation of the cytoplasmic domains of the <+ chains of the

T/><+ complex upon activation (99*.

As a ma7or conclusion$ the introduction of MAAA! emphasi%es the urgent need to



record the effects of cell-to-cell stimuli using real-time videos. We believe that MAAA!

holds huge scope that could be easily adapted to study various kinds of targets "such as

dots$ vesicles$ cells$ animals# based on various types of emitted signal$ however one

immediate application would be to compare our in vitro results to 0-photon imaging of

calcium indicator-loaded T cells migrating in lymph nodes (6+*$ (86*.

' !Materials and MethodsReagents and antiodies

0-Aminoethoxydiphenylborate "0-A5# ")= HM final concentration used for hybridomas$

0= HM for naive T cells# and thapsigargin ") HM final concentration# were purchased

from albiochem$ and ,onomycin "=.) Hg>mS$ final# from !igma. The 5D calcium

assay kit$ the antibody against <+I "clone )26-0))# "8.6 Hg>ml$ final#$ 0)) biot ")=

Hg>mS final# and the C0+.) anti-T/ @U : )-+ antibody ")= Hg>mS$ final# were supplied

by 5ecton <ickinson. The mitochondrion label$ Mitotracker red MD-/os$ and the

calcium indicators ,ndo-) AM$ Cluo-2 AM and Cura /ed AM were supplied by Sife

technologies "Molecular probes#. !treptavidin "6 Hg>mS# was supplied by Eackson

,mmunoresarch.2&+ "anti ,-Ak-&S#$ K4).6 "anti <2#$ and &);+.)8.+ "anti <26#

")= Hg>mS$ final# monoclonal antibodies were produced and purified in the lab from

hybridoma supernatants according to standard protocols.

Cell culture

+A; hybridoma T <2B cells are specific for hen egg lyso%yme peptide "&S# bound to

M& ,, ,-Ak molecules (9:*. These cells were cultured in /M, medium supplemented

with 6O C!$ ) mM sodium pyruvate and )= mM &epes.

'!-9 cells were cultured in <MM medium with 6O C!$ ) mM sodium pyruvate and

)= mM &epes. xperimental antigen-presenting cells "As# were generated by stablytransfecting '!-9 cells "Amaxa$ @ solution$ A=02# with plasmid c<3As coding for the

V chain of M& ,, and the U chain of M& ,, ,-A4 alone or covalently fused to a peptide

derived from &S "provided by <.A. @ignali# (28*. ells were sorted according to their

positivity to surface labeling by 2&+ antibodies "Cacsvantage$ 5ecton <ickinson#. A

monolayers were generated by seeding 6.6 )=2 cells into poly-S-Sysine-coated :-well

Sab-Tek chamber "3unc#.

!pleens and lymph nodes were recovered from 5A>E x +& non-transgenic mice and

+A; T/ transgenic mice (8+*$ (82*. After the extraction of cells onto nylon membrane in

<MM C)0 medium "Son%a#$ splenic erythrocytes were removed via 3&2l lysis. <2B

T cells were isolated by depleting the <2 negative cells according to manufacturer

instructions "<ynal Mouse <2 3egative ,solation 4it$ ,nvitrogen#.

Calcium imaging

The day before experiments$ cells were overnight serum starved in <MM-C)0 medium

supplemented with )O of 3utridoma-! "/oche#. Cor '!-9 transfected by plasmid

c<3As coding for the V and U chains of M& ,, ",-A4#$ &S peptide was added to the

culture medium the day before the experiment. oated surfaces were obtained by

incubation with the appropriate concentration of antibody 02 h before the experiment at

+9.



BD B*6 )=2 T cells per well were plated in ;8 well plates in )== HS of complete medium. ells

were loaded with 5< 5D diluted in )D dye loading solution "according to manufacturer

instructions# at )>)===e "∼) HM# for +A; cells ")>)888 for primary T cells i.e. ∼=.8

HM#. )== HS of this solution were dispatched to each well before incubation at +9

during ) hour "+= min for primary T cells# in the dark. ells were then washed twice in

&ankJs balanced salt solution "&5!!# &epes buffer containing ) mM calcium and

resuspended in the same medium. Cive wells were pooled "0.6 )=6 cells# and analy%ed

either by flow cytometry or microscopy. When mentioned$ cells were resuspended in

&5!! without a0B>Mg0B$ &epes ) mM. 0-A5 was added 7ust before recordings.

Additional experiments were performed by adding Cura /ed to the 5< 5D dye loading

buffer to a final concentration of 6.6 HM. Alternatively$ Cluo-2 AM was added to the

loading medium in the presence or in the absence of dye loading solution to a final

concentration of )= HM.

!ndo"+ AM0.6 )=8 cells were resuspended in 6== HS of complete &5!! buffer "&5!! without

calcium$ without Mgl0$ supplemented with ) mM al0$ ) mM Mgl0$ =.)O 5!A and

) mM &epes# with 6 HS ,ndo-) AM ")= HM# and incubated for += minutes at +9. ells

were washed twice in warm complete &5!! "+9# and ad7usted to the concentration of

6 )=6 cells>mS in complete &5!! before acquisition.

,low cytometry

ells were analy%ed on a S!/ , flow cytometer "5ecton <ickinson# with ell uest

software or S!/ ,, "for Cura /ed>5< 5D and ,ndo-) AM acquisitions# using the

CA!<iva software. 5D calcium indicator was observed over time on the CS) channel

with an excitation by an Argon laser 2:: nm and a 6+=>+= nm emission filter at +9$

maintained using a water bath. <ata analysis was performed with ClowEo software and

the median intensity of fluorescence was plotted vs. time after exclusion of dead cells and

cell debris.

Confocal microscopy

Movies were made on a Xeiss S!M 6)= Meta confocal microscope equipped with a +=

mW argon laser "06O output$ )O A'TC#. ictures were taken with a -Apochromat

2=Y>).0 water immersion ob7ective$ using the 2:: nm line of the argon laser$ &CT

?@>2:: dichroic mirror and a 6=6 nm long pass filter at +9$ maintained using a hot

plate. Time-lapse movies were composed of +== images "6)0Y6)0 pixels : bit 006

HmY006 Hm pinhole set to + airy units# taken every 9 seconds. Additional observationswere performed on an ?ltraview @oD erkin lmer spinning disk confocal microscope.

MAAACS analysis

All scripts$ including multiple target tracking "MTT# (+0*$ were developed under Matlab

"The Mathworks#. The source code of MTT$ deposited at the Agence pour la Protection

des Programmes$ n ,<<3.C/.==).09==0).=== !..0==:.===.+)0+=$ is freely available

for research purposes at httpN>>www.ciml.univ-mrs.fr>lab>he-marguet.htm. ell tracking

and automated analysis of cell signals with MAAA! can be done either in command

line "directly in Matlab# or using a graphical user interface "K?,#. While the K?, is more

intuitive it is limited to the analysis of a single acquisition whereas the command line

solution permits the sequential analysis of several video-acquisitions.

http://www.ciml.univ-mrs.fr/lab/he-marguet.htm

http://www.ciml.univ-mrs.fr/lab/he-marguet.htm



Cell trac#ing with MAAACSThe exhaustive analysis of cell signals depends on an efficient tracking in the video

acquisition stage$ meaning an excellent detection and reconnection of cells from frame to

frame. The MTT algorithm has already been optimi%ed to efficiently track single

molecule images. Thus$ the conversion of cells into single molecules needs to be

performed properly in order to allow the detection of all events in each frame. Mask

optimi%ation needs to be done for each and every cell type in order to mimic the cell

shape and si%e. Too small a mask could produce multiple detections for the same cell$

whereas a cell could be missed with too large a mask. The mask radius "in pixels# must

therefore be set by taking into consideration the mean cell radius "/cell# and the pixel

si%e on the image "Spix# at the magnification used during acquisition. The mask radius is

then set to /cell>Spix.

Cell signal analysis with MAAACSMAAA! automatically provides analytical parameters both on the fluorescence

intensity and on the tra7ectory of cells. Mainly$ it concerns their mean value$ the

persistence of high signal and the oscillation frequency. We have already developed the

parameters for the fluorescence intensity "namely the fluorescence amplitude$ the

response fraction and the number of bursts>min#. !imilarly$ this methodology has been

used to analy%e the cellular velocityN measured parameters were thus mean velocity "time-

averaged speed# and the mobile fraction "ratio of two phasesN the time when the speed is

greater than the threshold over the total time the cell is detected#.

,n addition$ we introduced an automatic method to classify the responses of activated

cells. This enabled us to classify response as Lmaintained when the response fraction

was higher than =.: and Lunique when the response fraction was lower than =.0 with a

single burst. ,n other cases$ calcium responses were defined as Loscillatory. We

considered the prototypic Lmaintained response as the T-cell calcium response upon

thapsigargin and the Lunique and transient response that upon thapsigargin with 0-A5.

The distributions of these cell responses were finally plotted as a pie-chart$ and revealed

the behavior of a cell upon a given stimulation and treatment.

Spectra analysis and in vitro -d determination

!pectra of 5< 5D and Cluo-2 AM were performed on the ary clipse

spectrofluorimeter "@arian#. In vitro 4d determinations were performed using the calcium

calibration kit "Sife technologies# according to the manufacturerJs instructions.

Statistical analysis

All statistical analyses and normality tests were performed using Kraphad rism 6.==.

To determine the normality of the data$ we performed a <JAgostino-earson normality

test. !ince not all our data were normally distributed$ we used a non-parametric statistical

test "two-tailed Mann-Whitney test with an alpha level of 6O#.

' !Supporting !nformation,igure S+

)valuation o! BD PBX as a reliable visible calcium inicator. "A# BD PBX



!luorescence. +A; T cells were loaded with Cluo-2 AM or 5< 5D with or without 5D

dye loading buffer. ells were seeded onto poly-S Sysine coated labtek wells at +9 and

imaged under a spinning confocal microscope. The same wells were imaged after += min.

"B# BD PBX !luorescence unless Fura%re is stable uner repetitive illuminations on

a con!ocal microscope. +A; T cells were loaded with 5< 5D or Cura /ed as indicated$

and seeded onto poly-S Sysine coated labtek wells at +9 and imaged under a confocal

microscope. Arrival of all cells in the observation field was synchroni%ed and the median

fluorescence value was expressed as a function of time. ,ndependent recordings were

analy%ed under similar experimental conditions. "C# )valuation o! calcium graient in

BD PBX loae cell hybriomas. +A; T cells were loaded with 5< 5D and

mitotracker red and imaged at +9 on a spinning disk confocal microscope equipped

with two simultaneous M< cameras. olocalisation plot were drawn. ells were

seeded onto non stimulating "3!# or stimulated "!# antibody coated labtek wells or onto

'!-9 ,-A4 A loaded or not with &S antigenic peptide. "D# Snapshots o! cells

loae concomitantly with BD PBX an mitotrac(er re in non%stimulating "9S# or

stimulate "S# conitions.

"T,C#

lick here for additional data file.").2M$ tif#

,igure S.

Diagram o! 'AAACS methoology. MAAA! methodology is mainly composed of

three ma7or data processing stepsN The first step "blue panel# concerns the image filtering$

the detection$ the tracking of cells and the automated normali%ation of the fluorescence

amplitude. The second one "optional$ green panel# allows the definition of optimi%ed

activation thresholds by comparing experimental conditions "such as As with or

without antigens#. The last step "pink panel# automatically calculates a series of analytical

parameters for each detected cell enabling the discrimination of activated and non-

activated cells and their categori%ation based on mode of response.

"!#

lick here for additional data file.":.;M$ eps#

,igure S/

Diversity o! calcium responses. !ingle cell fluorescence recordings with MAAA!

were performed on single +A; T cells stimulated by '!-9 ,-A4-&S "A# without or "B#

with the /A channel inhibitor 0-A5.

"!#

lick here for additional data file.").8M$ eps#

,igure S'

hreshol calculation o! calcium response. The activation threshold is systematically

evaluated both for +A; T cell hybridomas and naive T <2B cells and upon different way

of stimuli "antibody or with antigen-presenting cell A#. The probabilities of false alarm

"CA# and the probability of detection "<# are plotted as function of the activation

threshold "named as /' curves#. They are respectively estimated considering the

fluorescence amplitude of unactivated cells and activated cells upon each stimulation.

Then the optimal activation threshold is calculated by maximi%ing the score < x ")-

CA# "represented by a red dot#. The determination of the threshold is done without 0-

A5 "A# and in presence of 0-A5 "B#. "C# !ummary of the activation thresholds and

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784497/bin/pcbi.1003245.s001.tif

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their corresponding CA and < for +A; T cell hybridomas and naive T <2B cells$ and

upon different stimuli and treatments.

"!#

lick here for additional data file.").)M$ eps#,igure S0

elocity o! cell hybriomas upon various stimuli. "A# elocity BarcoingN The

velocity of each individual T cell "same cells "without 0-A5# as in !igure # was

concomitantly calculated with fluorescence intensity and displayed as a velocity barcode

"color coded on a log scale#. hapsigargin ";#< ells were stimulated with TK "nG+

independent recordings#. Anti CD& antiboy coate sur!ace< ells were seeded onto

anti <26 antibody coated Sab-tek chambers "nG)#. All tracked cells are represented on

the barcode. Anti C5 antiboy coate sur!ace< ells were seeded onto anti T/

antibody coated Sab-tek chambers "nG0#. Antigen presenting cell "APC#< ells were

seeded onto '!-9 experimental antigen presenting cells with peptides "see materials and

methods# "nG:#. "

B# !uperimposed tra7ectories of tracked +A; T cell hybridomasnormali%ed to their starting coordinates "each cell trace has been plotted in a randomly

chosen color#. ?pon stimulation with A$ the traces were plotted separately to

distinguish the behavior of unactivated from activated cells. "C# Analytical parameters

!or velocity uner i!!erent stimuliN The mean value "B>Z !M# for each analytical

parameter "velocity$ mobile fraction## is shown in red for each activated cell depicted as a

dot on each scatter plot.

"!#

lick here for additional data file."2.:M$ eps#

,igure S1

Diversity o! correlation between cell velocities an calcium in!lu$ in cell

populations. The velocity of a set of individual T cell was concomitantly color coded

with fluorescence intensity and displayed as a function of time. "A# anel of non-

activated or activated +A; T cell hybridomas. "B# anel of non-activated or activated

primary naive <2B +A; T cells.

"!#

lick here for additional data file.").8M$ eps#

,igure S2

C5AC channel activity in naive cells. "A# )!!ect o! )DA on the calcium responsewithin naive CD&: cells. 3aive <2B T cells were stimulated by anti <+ antibody

and calcium mobili%ation was measured by flow cytometry in the presence of increasing

concentrations of <TA. "B# ;lobal an intracellular mobili4ation o! calcium in naive

CD&: cells. The effect of 0-A5 on calcium mobili%ation was measured by flow

cytometry in naive <2B T cells after stimulation by thapsigargin. "C# Comparison o!

the e!!ects o! )DA an *%APB on intracellular calcium mobili4ation. The effects of

0-A5 and <TA were compared by flow cytometry in naive <2B T cells after

stimulation by anti <+ antibody.

"!#

lick here for additional data file."0.;M$ eps#













Tale S+

Summary o! all analytical parameters introuce an use by 'AAACS.

"!#

lick here for additional data file.").)M$ eps#

3ideo S+

5aw !luorescence movie o! cells loae with calcium inicator "'ethos#.

"M'@#

lick here for additional data file."+.9M$ mov#

3ideo S.

5aw !luorescence movie o! cells loae with calcium inicator in a -D

representation o! a 4one o! ieo S1.

"M'@#

lick here for additional data file."6.+M$ mov#

3ideo S/

'eian !iltere movie corresponing to ieo S1.

"M'@#

lick here for additional data file.";.+M$ mov#

3ideo S'

'eian !iltere movie corresponing to ieo S1 in a -D representation o! a 4one o!

vieo S-.

"M'@#

lick here for additional data file.":.8M$ mov#

3ideo S0

Single%molecule li(e movie corresponing to ieo S1.

"M'@#

lick here for additional data file."+.2M$ mov#3ideo S1

Single%molecule li(e movie corresponing to ieo S1 in a -D representation o! a

4one o! ieo S.

"M'@#

lick here for additional data file."6.:M$ mov#

3ideo S2

ra3ectories o! the cells !rom ieo S1 over each !rame as reconstructe by

'AAACS.


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"M'@#

lick here for additional data file."8.6M$ mov#

' !Ac#nowledgmentsWe thank Sionel osta and Cabien onchonaud for initiating this pro7ect$ and are

indebted to the imaging ",mag,mm# and cytometry ,MS core facilities for their technical

assistance and to hilippe ierre for providing the 2&+ hybridoma. We are grateful to

Marc 5a7[noff$ ![bastien Mailfert$ and Tomas% Trombik for their constructive comments.

We thank . Witty "Anglo!cribe# for editing the nglish.

' !,unding StatementA!aJs h< thesis was supported by fellowships from the Crench LMinist\re de la

/echerche and from the A/ "Association pour la /echerche contre le ancer#. M

was funded by C/M "Condation pour la recherche m[dicale$ Crance#. 5 was funded by

the Agence 3ationale de la /echerche "A3/ 5lanc-3anodigicode#. This work was also

funded by a specific grant from the Agence 3ationale de la /echerche "A3/-)=-5SA3-

)6=; and A3/-)=-,35!-=2-=) Crance 5io ,maging#. The funders had no role in study

design$ data collection and analysis$ decision to publish$ or preparation of the manuscript.

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0. Ceske ! "0==9# alcium signalling in lymphocyte activation and disease. 3at /ev

,mmunol 9N 8;=19=0 (ubMed*

+. Ceske !$ Kwack ]$ rakriya M$ !rikanth !$ uppel !&$ et al. "0==8# A mutation in

'rai) causes immune deficiency by abrogating /A channel function. 3ature 22)N

)9;1):6 (ubMed*2. Sewis /! "0==9# The molecular choreography of a store-operated calcium channel.

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http://www.ncbi.nlm.nih.gov/pubmed/18083096






http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2216848/























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