Anatomy of a flow cytometer

Fluidics

• Cells in suspension flow in single-file through an illuminated volume

Optics

• where they scatter light and emit fluorescence that is collected, filtered,

Electronics

• and converted to digital values that are stored on a computer

http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Fluorescence Detectors

If the cell is labeled with a

fluorescent probe which is

excited by the laser, the cell will

emit fluorescence as it passes

through the laser beam.

Increasing Fluorescence

http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Cell-by-Cell AnalysisCell-by-Cell Analysis

Fluorescent Plate Reader Flow Cytometer

Cell # FSC SSC FL1-H FL2-H FL3-H

1 447 315 4.45 6.55 8.03

2 557 277 12.30 12.30 10.13

3 582 267 6.21 9.56 15.86

4 413 390 17.75 17.79 19.46

5 249 399 6.04 5.53 18.25

6 542 351 11.34 27.41 20.03

…..

10,000 361 285 8.58 17.37 18.66

Data Display - Histograms

Frequency Distribution

1024 sets

offscale

data

Light ScatterLight Scatter Size

Shape

Granularity

Forward Scatter (FSC, FALS)

Most sensitive to size &

collection angle

Side Scatter (SSC, 90LS)

Most sensitive to granularity

http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Peripheral Blood Light Scatter

• Lymphocytes- small, little complexity

• Monoctyes-larger, more granular

• Neutrophils-large, lots of granules

Fluorochrome Comparisons

Comparative Intensities of CD8 Conjugates

FITCFITC PEPE ECDECD PC5PC5 PECy5.5PECy5.5 PC7PC7

APCAPC Alexa 700Alexa 700 APCAlexa 750APCAlexa 750

Pacific BluePacific Blue Pacific OrangePacific Orange

•Intrinsic CharacteristicsExtinction CoefficientQuantum YieldEmission Spectral Overlap

•Instrument OpticsFiltersPMT SensitivityLaser wavelength & power

Fluorochrome Selection

Low density expression needs a bright fluorochrome.

E-Cadherin FITC

FITC isotype

18%

E-Cadherin PE

PE isotype

71%

CompensationCorrection for spectral overlap

FITC & PE Emission Spectra

FITC only sample

FITC spillover

compensated

CompensationRules of the Road

1. Controls need to be as bright or brighter than

any sample that the compensation will be

applied to.

2. Background fluorescence should be the same

for the positive and the negative control.

3. Compensation controls MUST match the exact

experimental fluorochrome.

Analysis- 6 colors

Analysis 6 colors, 15 dot plots

Multiparameter Analysis – 6 colorsMultiparameter Analysis – 6 colors

• Data filtering to select

populations of interest

• Color events based on

gates to visualize multi-

parameter data

Cell Sorting

Sort Purification

Subset of interest = 1%

Sort rate = 20,000 total cells/s

Collection rate = 200 cells/s

Or ~83 min for 1 million cells

presort postsort

Walter and Eliza Hall Institute Flow

Cytometryhttp://www.wehi.edu.au/cytometry/flowintrol.html

Sort into any size plate with any number of cells per well

Plate SortsPlate Sorts

Tips for better cell sorting

• Avoid cell aggregates

• Use a viability stain

• Bright markers yield higher purity

• Avoid compensation as much as possible

J. Dow, NC State Vet Med

Our favorite websites

Compensation, Mario Roederer

http://www.drmr.com/compensation/

Flow Cytometry- A Basic Introduction, Michael Ormerod

http://www.flowbook.denovosoftware.com/site/introtoflowormerod.shtml

Life Technologies

Fluorescence Tutorials

Spectraviewer

http://www.lifetechnologies.com/us/en/home/support/tutorials.html

University of Chicago Flow Cytometry Blog Spot, Ryan Duggan

10 steps to a successful flow cytometry experiment

Antibody titrations

What is MFI?

http://www.ucflow.blogspot.com

Submitted QuestionsI’m new to the Gallios. How do I choose voltage settings?

• Use the minimum voltage that resolves a dim population

• Determine using the CV of unstained cells or signal:noise of stained cells

• Brightest positive signals must be on-scale

• PMT settings should be balanced to permit compensation

PMT Filter Suggested

starting

voltage

1 525/40 400

2 575/30 400

3 620/30 450

4 695/30 500

5 755LP 550

6 660/30 500

7 725/20 500

8 755LP 550

9 450/50 400

10 550/40 450

Submitted QuestionsWhy am I having trouble analyzing Gallios data with FlowJo?

• Scaling problems

• Compensation problems

Scaling problems seem to be fixable, but compensation should be done

on the Gallios

Kaluza for Gallios acquisition data (FCS3.1) should work with

FlowJo Mac version 8 or better

FlowJo X

Submitted QuestionsWhat do I need to think about for quality control of flow assays?

Instrument QC

• Alignment check- %CV

• PMT standardization- adjust voltage to place reference bead in same channel

• Check compensation

Antibody QC

• Titer new lot of antibodies

• Check for lot-to-lot consistency

• Check compensation

Reagent QC

• Validate new lot of reagents (Buffers, lyse reagents)