CellutionsVol 1: 2012The Newsletter for

Cell Biology Researchers

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

Take the CHOre out of Protein Expression

Rapid Analysis of Human Adipose-Derived Stem Cells and 3T3-L1 Differentiation Towards Adipocytes Using the Scepter™ 2.0 Cell Counter Page 6

Isolation and Characterization of Human Bone-Marrow-Derived Mesenchymal Stem Cells Page 12

Inhibition of TGFβRI Reduces Levels of Phosphorylated Smads: Elucidation of TGFβ Signaling Using a Multianalyte Immunoassay and Chemical Genetics Page 14

Identification and Functional Characterization of Natural Killer Cells Using Flow Cytometry of Autophagosome Formation Page 18

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Efficient and Scalable Protein Expression in Suspension CHO Cells Using the NovaCHOice® Transfection System Page 3

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PRODUCT HIGHLIGHT

Goodbye, Bradford Assays! Drive your research forward with IR-based quantitation.With the Direct Detect™ system, the first infrared (IR)-based biomolecular quantitation system, there’s no sample prep, messy cuvettes or waste—with one-time standard curves. The Direct Detect™ system distinguishes proteins and peptides from interfering sample components, such as lipids. Now you can achieve truly accurate results without the pitfalls of colorimetric assays, even for most lysates and complex samples.

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Quit Assays Forever — Quantitate Directly.Learn more at: www.millipore.com/DirectDetect

Accurate IR-based protein quantitation in a lipid-rich lysate. The most intense regions of lipid absorbance are distinct from the protein’s Amide I signal.

3

IntroductionProtein expression in mammalian cells is the method of

choice for the production of bioactive proteins displaying

appropriate post-translational modifications. A convenient

approach to obtain bioactive proteins and shorten the

evaluation cycles is to use transient transfection. One of the

preferred cells systems for production of bioactive proteins is

Chinese Hamster Ovary (CHO) cells that have been adapted

for growth in suspension. However, suspension CHO (CHO-S)

cells tend to be refractory to transfection using many of

the commonly-used transfection reagents. Here we present

data on the use of the NovaCHOice® transfection system,

consisting of a core reagent and an optional booster, to

obtain high levels of protein expression in CHO-S cells. Our

data indicate that use of NovaCHOice® transfection system

results in high transfection efficiency and high levels of

protein expression within 24 h. Our data further indicate that

by 48 h and 72 h the proportion of high-expressing cells

is significantly increased with very low cellular toxicity. In

addition, no medium changes are required post-transfection,

thus reducing cell losses and hands-on time. In summary,

the NovaCHOice® transfection system is well-suited for

transient and stable transfections in CHO-S cells, due to its

high transfection efficiency and low toxicity.

Materials and MethodsProtein expression in CHO-S cells was evaluated using

transfection with plasmids expressing either Green

Fluorescent Protein (GFP) or human Secreted Alkaline

Phosphatase (SEAP). CHO-S cells were transfected using

the NovaCHOice® Transfection Kit in 15 mL cultures in a

shaker flask unless otherwise indicated. The day prior to

transfection, the cells were passaged at 0.5 x 106 cells/mL

and diluted to a concentration of 1x106 cells/mL the day

of transfection.

All cell concentrations were determined using the Scepter™

handheld, automated cell counter. For 15 mL transfection,

15 µg DNA, 15 µL NovaCHOice® Transfection Reagent and

7.5 µL NovaCHOice® Booster Reagent were mixed in 1500 µL

OptiPRO™ medium (Life Technologies; 1:1:0.5 ratio). DNA

was added into the prepared tube with culture medium,

followed by the transfection reagent then the booster

reagent. and the mixture was mixed gently and allowed to

incubate at room temperature for 15 minutes before it was

added dropwise to the flask of cells.

SEAP expression was determined at 24, 48 and 72 h

post-transfection by removing 50 µL of the culture at

the corresponding time point, centrifuging the cells and

collecting the supernatant. The supertantant was kept at

-20 °C until assayed. Supernatants were assayed using the

SEAPorter™ kit (Imgenex, Cat. No. 10055K).

Expression of GFP was evaluated by fluorescent imaging and

by flow cytometry. For fluorescence imaging, 500 µL of the

culture was removed at 24, 48 and 72 h post-transfection

and plated onto poly-L-lysine-coated 24-well plates. An

additional 500 µL of sample was also removed from each

flask at 24, 48 and 72 h for flow cytometry analysis.

To determine the scalability of transfection with the

NovaCHOice® Transfection Kit, 10 mL, 100 mL and 1000

mL cultures were transfected with SEAP-encoding plasmid

DNA (SEAP pDNA) as described above. Culture samples were

collected at 24, 48 and 72 h post-transfection.

Efficient and Scalable Protein Expression in Suspension CHO Cells Using the NovaCHOice® Transfection SystemJackie Stupack, Alok Tomar and Adrian VilaltaEMD Millipore Corporation

4

Figure 3. Fluorescence imaging indicates high transfection efficiency. Images were acquired at 24 h, 48 h and 72 h post-transfection using an inverted fluorescent microscope (Zeiss Axiovert 200). Cells expressing GFP were imaged using FITC channel (Ex. 490, Em. 525) and merged with the brightfield image using ImageJ, version 1.4.3.67.

Figure 1. High levels of SEAP expression is evident 24 h post-transfection. Levels of SEAP secreted into the growth medium reached 1050 ng/mL 24 h after transfection; an increase of close to 20% in levels of secreted SEAP were evident 72 h post-transfection. Error bars indicate standard deviation.

1250

24 h 48 h 72 h

SEAP

(ng/

mL) 1150

1000

1200

1100

1050

950

field expressed the fluorescent marker. In addition, cell

morphology as visualized during imaging indicated low

toxicity after transfection.

These observations were supported by the flow cytometric

analysis (Figure 4), which showed that 90% of the cells

were positive for GFP expression 24 h after transfection

and close to 99% by 48 h.

Figure 2. The NovaCHOice® Transfection Kit allows for scalable expression of SEAP in CHO-S systems. Levels of SEAP in the culture medium were determined as described in the Materials and Methods section at the indicated time points. Total SEAP produced at each scale and time point closely followed the 10-fold step increase in culture size.

10000

24 h 48 h 72 h

SEAP

(µg)

100

1000

10

1

10 mL100 mL1000 mL

Bright Field

24 h

GFP

48 h

72 h

Merge

ResultsHigh levels of SEAP expression (~1,000 ng/mL) was

observed 24 h post-transfection with a maximum of

(~1,200 ng/mL) after 48 h (Figure 1).

Transfection scalability data are shown in Figure 2. SEAP

expression levels increased proportionally to volume at

all time points. This observation was consistent with

the fluorescence imaging and flow cytometric GFP

expression data. Fluorescence imaging data (Figure 3)

showed that a high proportion of the cells were expressing

the GFP marker at 24 h; by 48 h nearly all cells in the

5

Description Catalogue No.

NovaCHOice® Transfection Kit 72622-3 and 72622-4

Scepter™ 2.0 Handheld Automated Cell Counter PHCC20060

Available from www.millipore.com.

FEATURED PRODUCTS

Figure 4. Transfection of CHO-S cells using NovaCHOice® Transfection Kit results in high transfection efficiency. Flow cytometry data for GFP-expressing cells are shown for the time points indicated. A high proportion of cells was positive for GFP expression 24 h post-transfection.

DiscussionThe data presented here demonstrate that the NovaCHOice® transfection system is

well-suited for rapid, high levels of protein expression in suspension CHO cells. Maximum

levels of expression of both secreted (SEAP) and intracellular (GFP) reporters are obtained

24-48 h after transfection. Both fluorescence imaging and flow cytometric analysis

indicate that transfection efficiencies approach 99% 48 h post-transfection; in addition,

flow cytometric data show a high proportion of high expressing cells. Although only

transient transfection data are presented here, it would be expected that the NovaCHOice®

transfection system would be attractive as a first step in the production of stably

expressing lines due to its high transfection efficiency and low toxicity. Importantly,

transfections are scalable from 10 mL to 1 L, which makes the NovaCHOice® transfection

system attractive for scientists interested in producing large quantities

of protein by transient transfection.

M1

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For protein research as well as for the development of

large-molecule biotherapeutics, it is crucial to produce

and purify sufficient quantities of functionally active,

highly pure, recombinant protein. Using the same batch of

recombinant protein across all studies can help eliminate

equivocal results generated by lot-to-lot variability in

protein activity and sample composition. Therefore, it is

highly desirable to obtain maximum yield from each batch

of cells expressing the recombinant protein.

By enabling researchers to quickly obtain high yields of

recombinant protein in CHO-S cells, the NovaCHOice®

transfection system has the potential to accelerate protein

research and maximize the information obtained from

every experiment.

Negative Control

48 h

24 h 72 h

6

Rapid Analysis of Human Adipose-Derived Stem Cells and 3T3-L1 Differentiation Towards Adipocytes Using the Scepter™ 2.0 Cell Counter

Wenying Zhang, Mark Santos, and Matthew HsuEMD Millipore Corporation

IntroductionObesity is a prevalent health hazard in many industrialized nations, and its occurrence is closely associated with a number of pathological disorders, such as non-insulin dependent diabetes, hypertension, cancer, and atherosclerosis1. Because dysregulated adipogenesis and fat cell functions play central roles in these disorders, there has been a surge in efforts to understand the cellular and molecular mechanisms of adipocyte differentiation. Adipocytes are derived from multipotent human mesenchymal stem cells (MSCs), which are found within the bone marrow stroma. MSCs have the potential to differentiate towards many different lineages of mesenchymal tissues including bone, cartilage, muscle, and fat2.

Most of our knowledge about adipogenesis comes from in vitro studies using preadipocyte culture systems1. Human Adipose-Derived Cells (ADSCs) are adult stem cells isolated from human lipoaspirates in adipose tissue, and like MSCs, differentiate in vitro toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages when treated with established lineage-specific factors. Given the multi-lineage differentiation potential of ADSCs, these cells are phenotypically similar to MSCs4, 5, thus providing researchers an ideal system for studying adipogenesis. 3T3-L1 cells are undifferentiated fibroblast-like preadipocytes that are also frequently employed in most adipogenesis studies. These were clonally isolated from Swiss 3T3 cells derived from disaggregated 17- to 19-day-old mouse embryos.

Unique characteristics of adipocyte differentiation include dramatic changes in cell size, shape, membrane potential, metabolic activity, and responsiveness to signals6.

When ADSCs and 3T3-L1 cells commit to the adipocytic lineage, lipid-rich vacuoles accumulate within these cells. These vacuoles continue to develop over time and expand, eventually filling and enlarging the cellular diameter. We hypothesized that using the Scepter™ cell counter to rapidly assess size distributions of cellular populations would provide a quick, simple method for tracking adipocyte differentiation.

The Scepter™ cell counter captures the ease of automated instrumentation and accuracy of impedance-based counting using the Coulter principle in an affordable, handheld format. The instrumentation has been collapsed into a device the size of a pipette, and uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display in the form of a histogram. The 40 µm- and 60 µm-aperture sensor tips are engineered with a microfabricated, sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution, respectively. The histogram output provides a quick snapshot of cell size and density.

This study outlines a method for tracking adipogenic differentiation of ADSCs and 3T3-L1 cells and subsequent sample analysis using the Scepter™ cell counter to measure these changes. We have employed Scepter™ technology for determining cell size and volume, as well as lipid vacuole staining using Oil Red O as a cross-validation histology screen to investigate the relationship between cell differentiation and cell size changes. Although the Scepter™ cell counter was intended primarily as a cell counting device, we demonstrate how this cell counter can also function as a reliable tool to track phenotypic change.

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Adipogenesis Differentiation: Adipose-Derived Stem Cells (ADSCs)

Adipose-Derived Stem Cells were plated at a density of 50,000 cells per well into gelatin (EMD Millipore Cat. No. ES-006-B) -coated 12-well culture dishes with 1 mL medium per well. Cells were incubated at 37 °C in a 5% CO2 humidified incubator for 4 days. When the cells were 100% confluent, medium was aspirated from each well and replaced with 1 mL Adipogenesis Induction Medium (EMD Millipore Cat. No. SCR020). This medium change was set as differentiation day 1.

Medium was replaced with fresh Adipogenesis Induction Medium every 2-3 days for 14 days. After 0, 7 and 14 days of differentiation, adipocytes were fixed and the lipid droplets stained with Oil Red O Solution. Lipid droplets could be detected by microscopic examination as early as 5 days into the differentiation period.

Adipogenesis Differentiation: 3T3-L1 cells

3T3-L1 cells (ATCC® Cat. No. CL-173TM) were plated in 10% calf serum/DMEM Medium at a density of 50,000 cells per well into 12-well culture dishes with 2 mL medium per well. Cells were incubated at 37 °C in a 10% CO2 humidified incubator for 4 days.

When the cells were 100% confluent, the medium was aspirated from each well and replaced with 2 mL MDI Induction Medium. This medium change was set as differentiation day 0.

At differentiation day 2, the medium was replaced with Insulin Medium. The medium tended towards greater viscosity as free fatty acids were produced by the cells and secreted into the medium. At differentiation day 4, the medium was replaced with MDI Induction Medium. At differentiation day 6, the medium was replaced with Insulin Medium. At differentiation day 8, the medium was replaced with 10% FBS/DMEM.

Cells were subsequently fed with 10% FBS/DMEM every two days. After differentiation, >90% of cells were mature adipocytes with accumulated fat droplets. After 7 and 14 days of differentiation, adipocytes were fixed and the lipid droplets stained with Oil Red O Solution.

Materials and MethodsWe implemented a previously validated protocol for proper maintenance and treatment of preadipocyte cultures to differentiate into terminally differentiated adipocytes. We encountered slight variations in culture conditions for each cell type, but measurement of lipid vacuoles by Oil Red O staining remained the same for both cultures.

Media Formulations

10% Calf Serum/DMEM• Calf Serum (Gibco Cat. No.16170)• 100 Units/ml Penicillin and 100 µg/ml Streptomycin

(EMD Millipore Cat. No. TMS-AB2-C)• DMEM (Gibco Cat. No. 11965-084: high glucose, with

L-glutamine, with pyroxidine HCl, without sodium pyruvate)

10% FBS/DMEM• Fetal Bovine Serum (EMD Millipore Cat. No. ES-009-B)• 100 Units/ml Penicillin and 100 µg/ml Streptomycin

(EMD Millipore Cat. No. TMS-AB2-C)• DMEM (Gibco Cat. No. 11965-084: high glucose, with

L-glutamine, with pyroxidine HCl, without sodium pyruvate)

MDI Induction Medium • 0.5 µm Isobutylmethylxanthine (IBMX;

Sigma Cat. No. I-7018)• 1 µm Dexamethasone (Sigma Cat. No. D-4902)• 10 µg/ml Bovine Insulin (Gemini Cat. No. 700-112P)• 10 % FBS/DMEM

Insulin Medium• 10 µg/ml Bovine Insulin (Gemini Cat. No. 700-112P)• 10 % FBS/DMEM

8

Oil Red O Staining

Oil Red O is a fat-soluble dye used for staining neutral triglycerides and lipids. This reagent is commonly used to identify exogenous or endogenous lipid deposits in cells. Here, we used Oil Red O for the detection of lipid-rich vacuoles commonly found in adipocytes. Prior to cell staining, cells (preadipocytes, 7 day differentiation, or 14 differentiation) were plated into a 24-well tissue culture plate.

Medium was first carefully aspirated from the cells. Adipocytes were fixed by incubating in 4% paraformaldehyde for 30-40 minutes at room temperature. The fixative was aspirated and cells were rinsed three times (5-10 minutes each) with 1X phosphate-buffered saline (PBS). The PBS was aspirated and the cells rinsed twice with water.

The water was aspirated and enough Oil Red O Solution added to cover the wells (500 µL to 1 mL per well in a 24-well plate). The cells were incubated at room temperature for 50 minutes. The Oil Red O Solution was removed and wells washed three times with 1 mL water.

Cell nuclei were stained with with Hematoxylin Solution (0.5 mL volume) for 5 to 15 minutes. Adipocytes containing lipid droplets were stained red by the Oil Red O solution while the cell nuclei were stained black/blue from the hematoxylin.

Scepter™ Cell Counting

The Scepter™ cell counter was used to count samples following the detailed on-screen instructions for each step of the counting process. Briefly, the user attaches a 60 µm sensor tip, depresses the plunger, submerges the sensor into the sample, then releases the plunger drawing the

cell suspension into the sensor. The Scepter™ cell counter detects each cell passing through the sensor’s aperture, calculates cell concentration, and displays a size-based histogram as a function of cell diameter or volume on its screen. Scepter™ 2.1 software was then used to upload test files from the device and perform subsequent data analysis to determine cell size and relative cell frequencies for the preadipocyte cultures (ADSCs and 3T3-L1 cells). The initiation of differentiation culture conditions was noted to mark day 0, differentiation at day 7, and differentiation at day 14.

ResultsPreadipocyte cultures were treated with established lineage-specific factors for fourteen (14) days to drive adipogenesis and formation of terminally differentiated adipocytes. Both ADSCs and 3T3-L1 cells were used in this study since both cell types have been commonly utilized when performing in vitro analysis of adipogenesis. Moreover, both cell types have been well characterized and contain the same cellular machinery as seen in multipotent mesenchymal stem cells.

Adipose-Derived Stem Cell Differentiation Analysis

ADSCs were induced to differentiate towards adipocytes. Samples were gently trypsinized and analyzed using the Scepter™ cell counter. Undifferentiated cells (Blue) exhibited a mean cell diameter of 14.9 µm; cells differentiated for 7 days (Purple) had a mean cell diameter of 18.4 µm; cells differentiated for 14 days (Red) had a mean cell diameter of 21.1 µm (Figures 1 and 5). Concurrently, the same samples from different wells were stained with Oil Red O (which stains lipid vacuoles) and counterstained with hematoxylin (Figure 2). Histology images were captured using a Leica DMI 6000, 40X objective lens.

Figure 1. Preadipocytes (ADSCs) can be distinguished from differentiated adipocytes based on cell size. Using a 60 µm sensor, the Scepter™ cell counter enabled the discrimination of cell types based on size, with high resolution. ADSCs were measured at three key time points: Day 0 (control), seven (7) days after exposure to differentiation conditions, and fourteen (14) days differentiated. As indicated by the histogram data, cells gradually increased in size from 15 to 21 µm over the fourteen-day differentiation.

Undifferentiated ADSCs

7 Day Differentiation

14 Day Differentiation

9

A. Undifferentiated (ADSCs)

B. 7 Day Differentiation

C. 14 Day Differentiation

A. Undifferentiated (3T3-L1)

B. 7 Day Differentiation

C. 14 Day Differentiation

3T3-L1 Cell Differentiation Analysis

In a similar fashion, 3T3-L1 cells were induced to differentiate towards adipocytes. Samples were gently trypsinized and analyzed using the Scepter™ cell counter. Undifferentiated cells (Blue) exhibited a mean cell diameter of 15.4 µm; cells differentiated for 7 days (Purple) had a mean cell diameter of 18.8 µm; and cells differentiated for 14 days (Red) had a mean cell diameter of 20.3 µm (Figures 3 and 5). Concurrently, the same samples from different wells were stained with Oil Red O and counterstained with hematoxylin (Figure 4). Histology images were captured using a Leica DMI 6000, 40X objective lens.

Figure 4. Oil Red O staining was used to measure the lipid content of adipocytes obtained from differentiated 3T3-L1 cells. 3T3-L1 cells and differentiated samples taken from the same critical time points (7 and 14 days) were stained with Oil Red O. Again, a gradual increase in lipid content between days 7 and 14 during adipogenesis correlated with the cell size measurements determined by the Scepter™ cell counter.

Figure 3. 3T3-L1 cell differentiation into adipocytes could be clearly identified based on changes in cell size. Using a 60 µm sensor, the Scepter™ cell counter enabled the discrimination of cell types based on size, with high resolution. Like ADSCs, 3T3-L1 cells were measured at three key time points: Day 0 (control), seven (7) days after exposure to differentiation conditions, and fourteen (14) days differentiated. As indicated by the histogram data, cells gradually increased in size from 15 to 21 µm over the fourteen-day differentiation.

Figure 2. Oil Red O staining was used to measure the increased lipid content of adipocytes compared to undifferentiated cells. Undifferentiated ADSCs and differentiated samples taken from the same critical time points (7 and 14 days) were stained with Oil Red O. Oil Red O stains lipid vacuoles, enabling visual quantification of lipid content in cells by microscopy (40X objective). As seen, increased lipid content, which is common for mature adipocytes, correlates with the cell size measurements determined by Scepter™ counting.

Undifferentiated 3T3-L1

7 Day Differentiation

14 Day Differentiation

10

Using the Scepter™ cell counter to determine cell size and distribution of the cell cultures, an increase in cell size relative to the number of days during cell differentiation was accurately determined. Each experiment was performed on three separate occasions to validate our findings, and, each time, cross-validation using Oil Red O staining confirmed these results. Small standard deviations of the mean measured diameter of three cell samples demonstrated the precision of the Scepter™ cell counter (Table 1).

Both ADSCs and 3T3-L1 cells were performed in triplicate assays (three independent experiments, plus three samples per experiment) and standard deviation values were determined. These data demonstrated the precision of the Scepter™ cell counter for performing cell size determination across many test samples.

Experiment #1 Experiment #2 Experiment #3

Scepter™ values (ADSCs) n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3 Average diameter (μm) Standard Deviation

Day 0 14.8 14.9 14.9 15.6 15.9 15.7 14.8 14.9 14.9 15.2 0.4

Day 7 18.3 18.4 18.5 19.8 19.9 20.0 19.6 19.4 19.5 19.3 0.7

Day 14 20.4 20.3 19.7 23.3 20.0 19.9 21.2 21.0 21.2 20.8 1.1

Scepter™ values (3T3-L1) n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3 Average diameter (μm) Standard Deviation

Day 0 15.3 15.6 14.9 15.1 15.1 14.9 15.6 15.4 15.4 15.3 0.3

Day 7 18.6 18.8 18.6 18.5 18.6 18.7 18.6 18.9 19.0 18.7 0.2

Day 14 20.0 20.1 20.2 19.9 19.8 19.6 20.1 20.3 20.5 20.1 0.3

Table 1. High precision of Scepter™ cell counting. Comparison of cell diameter measurements for both ADSC and 3T3-L1 cells.

0

5

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Adipogenesis (Preadipocyte Differentiation)

Day 0 Day 0Day 14 Day 14Day 7 Day 7

Figure 5. Cell diameters of ADSCs and 3T3-L1 cells undergo comparable increases during adipocyte differentiation (adipogenesis) as measured using the Scepter™ cell counter

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ConclusionsMesenchymal stem cell research and understanding its relationship to adipogenesis remains an important area for research with the realization that society may be in the early stages of a global wave of obesity. The ability to isolate, expand, and direct the in vitro differentiation of ADSCs and 3T3-L1 cells to particular lineages provides the opportunity to study events associated with commitment and differentiation, and, in the case of this study, the development of terminally differentiated adipocytes.

Adipocytes provide a safe place to store lipids. It has been well documented that during the process of adipogenesis, preadipocytes convert to a spherical shape and accumulates lipid droplets, resulting in an increase in cell diameter as these lipid-rich vacuoles fill and grow within the cell. The Scepter™ cell counter provides a rapid, easy, and inexpensive method for assessing ADSC and 3T3-L1 differentiation in culture. This handheld, automated cell counter delivers precise, reliable cell size measurements which can provide the researcher a quick snapshot of the differentiation status and the success of their differentiations in culture. As a result, researchers can fine-tune culture conditions and adjust analysis parameters to maximize information obtained from each experiment, make better experimental decisions, and ultimately, enjoy more productive research.

Description Quantity Catalogue No.

Scepter™ 2.0 Handheld Automated Cell Counter

with 40 µm Scepter™ Sensors (50 Pack) 1 PHCC20040

with 60 µm Scepter™ Sensors (50 Pack) 1 PHCC20060

Includes:

Scepter™ Cell Counter 1

Downloadable Scepter™ Software 1

O-Rings 2

Scepter™ Test Beads 1 PHCCBEADS

Scepter™ USB Cable 1 PHCCCABLE

Scepter™ Sensors, 60 µm 50 PHCC60050

500 PHCC60500

Scepter™ Sensors, 40 µm 50 PHCC40050

500 PHCC40500

Universal Power Adapters 1 PHCCP0WER

Scepter™ O-Ring Kit, includes 2 O-rings and 1 filter cover 1 PHCC0CLIP

Reagents/Kits

EmbryoMax® 0.1% Gelatin Solution 1/Pk ES-006-B

Mesenchymal Adipogenesis Kit 1/Kit SCR020

FBS ES-009-B

Penicillin/Streptomycin TMS-AB2-C

Available from www.millipore.com.

RELATED PRODUCTS

References1. Ogden, C.L., et. al. (2006). Prevalence of overweight and obesity

in the United States, 1999-2004. JAMA.;295(13):1549-55.2. Pittenger, M.F., et. al. (1999). Multilineage potential of adult

human mesenchymal stem cells. Science;284(5411):143-7.3. Gregoire, F.M., et. al. (1998). Understanding adipocyte

differentiation. Physiol Rev.; 78(3):783-809.4. Zuk, P.A., et. al. (2002). Human adipose tissue is a source of

multipotent stem cells. Mol Biol Cell.;13(12):4279-95.5. Zuk, P.A., et. al. (2001). Multilineage cells from human

adipose tissue: implications for cell-based therapies. Tissue Eng.;7(2):211-28.

6. Rosen, E.D., et. al. (2006). Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol.;7(12):885-96.

12

AbstractMesenchymal stem cells derived from bone marrow have

become an attractive model cell system for the in vitro and

in vivo study of disease states such as osteoporosis, obesity,

and arthritis. We have isolated mesenchymal stem cells

(MSCs) from the bone marrow aspirate of a single human

donor. Human bone-marrow-derived MSCs express the

mesenchymal stem cell markers STR0-1, CD44, and CD90,

and do not express hematopoietic markers CD14 and CD19.

Human bone-marrow-derived MSCs can be expanded for

18 passages and are multipotent, possessing the ability to

differentiate into adipocytes and osteocytes.

IntroductionThe bone marrow stroma consists of heterogeneous

populations of endothelial cells, adipocytes, osteocytes,

and fibroblast cells, along with a mixture of multipotent

progenitor cells. Recently, two distinct multipotent cellular

populations have been discovered within the bone marrow:

a hematopoietic stem cell population responsible for

replenishing blood cells, and a mesenchymal stem cell

population responsible for regenerating non-hematopoietic

cell elements within the bone marrow. MSCs have the ability

to self-renew and differentiate into adipose, bone, and

cartilage tissues; these characteristics make them extremely

useful in the study of numerous cellular processes including

bone, fat and cartilage formation and degradation.

Using a colony forming unit (CFU) isolation technique,

we have established a viable low passage (<p5), human

bone-marrow-derived MSC cell line capable of rapid

in vitro expansion (up to passage 18) and multipotent

differentiation. These human MSC cells retain a normal

karyotype and differentiation capacity up to passage 18

in serum-containing media. The cells provide a reliable,

functional, and validated source of human bone-marrow-

derived MSCs for researchers.

MethodsBone marrow aspirates from the iliac crest of a single normal

human donor were cultured in serum-containing growth

media plus 4 ng/mL b-FGF and allowed to adhere for 3

days on a gelatin-coated T 175 tissue-culture-treated flask.

After three days, non-adherent cells were washed away and

adherent, fibroblast-like cells were expanded in serum-

containing media plus 4 ng/mL b-FGF over multiple weeks

until ready for harvesting.

For marker expression analysis, cells were plated onto 0.1%

gelatin-coated, 8-well chamber slides at 2.5 x 104 cells per

well and allowed to grow for 2 days in growth media. Cells

were fixed with 4% paraformaldehyde and stained with

antibodies from EMD Millipore’s human mesenchymal stem

cell characterization kit. All antibodies were diluted 1:500.

Cells were differentiated to adipocytes and osteocytes

following EMD Millipore’s mesenchymal stem cells

adipogenesis and osteogenesis kits, respectively.

Isolation and Characterization of Human Bone-Marrow-Derived Mesenchymal Stem CellsNick Asbrock and Vi Chu EMD Millipore Corporation

13

ResultsBone-marrow-derived mesenchymal stem cells display

fibroblast-like morphology in monolayer culture (Figure

1A and 1B) and stain positively for mesenchymal stem cell

markers CD44 (Figure 2A), CD90 (2B), and STRO-1 (2C).

They also lack expression of hematopoietic markers CD19

and CD14 (data not shown). Cells reached 80% confluency

(Figure 1B) within two days after thawing and continued to

actively divide up to passage 18 while retaining a normal

karyotype (Figure 1C). Cytogenetic analysis (performed by

Cell Line Genetics) on twenty G-banded metaphase cells

demonstrated an apparently normal male karyotype (46, XY)

and no abnormal cells were detected.

Human bone-marrow derived MSCs are multipotent and

have the capacity to differentiate into lipid-containing

adipocytes (Figure 3A-B) or calcium-secreting and alkaline-

phosphatase-expressing osteocytes (Figure 3D-F), following

defined differentiation protocols.

ConclusionWe have developed a new, low passage, human bone-

marrow-derived mesenchymal stem cell line which possesses

the correct mesenchymal stem cell morphology, marker

expression, and multipotent differentiation capabilities.

These cells provide a convenient and fully validated source of

human mesenchymal stem cells for researchers interested in

mesenchymal stem cell biology.

A. B. C.

A. B. C.

A.

D.

B.

E.

C.

F.

Figure 1. Phase contrast images of human mesenchymal stem cells one (A) and two (B) days after thawing. Cells possess an apparently normal karyotype (C).

Figure 2. Human mesenchymal stem cells express H-CAM (CD44) (A), THY-1 (CD90) (B), and STRO-1 (C). Nuclei were visualized with DAPI (blue). Expression of hematopoietic stem cell markers CD19 and CD14 and endothelial marker CD146 were not observed (data not shown).

Figure 3. Human mesenchymal stem cells are multipotent, as demonstrated using EMD Millipore’s mesenchymal stem cell adipogenesis (A-C) and osteogenesis (D-F) kits. Mesenchymal stem cells differentiated after 21 days to mature adipocytes as indicated by the accumulation of lipid vacuoles that stain with oil-red-O (A, 10x magnification; B, 20x magnification). Control untreated cells did not contain any lipid droplets (C). Using the osteogenesis kit, stem cells differentiated to an osteocyte lineage as indicated by alizarin red S staining (D) and alkaline phosphatase (E, 4x magnification; F, 20x magnification) staining. Alizarin red S staining demonstrates mineral deposition throughout the culture.

Description Catalogue No.

Human Mesenchymal Stem Cell Kit (>106 cells + 500 mL media) SCR108

Human Mesenchymal Stem Cell Characterization Kit SCR067

Mesenchymal Stem Cells Adipogenesis Characterization Kit SCR020

Mesenchymal Stem Cells Osteogenesis Characterization Kit SCR028

Mesenchymal Stem Cell Expansion Medium, 1X SCM015

Fibroblast Growth Factor basic (FGF-b), human recombinant GF003

Available from www.millipore.com.

RELATED PRODUCTS

14

IntroductionThe transforming growth factor beta (TGFβ) signaling

pathway plays a crucial role in critical cellular processes

including cell proliferation, differentiation, homeostasis,

and apoptosis. Dysregulation has been implicated in as the

cause of carcinogenesis and tumor progression in a variety

of cancers.

Binding of TGFβ to TGFβ receptor II (TGFβ RII) initiates

signaling in the TGFβ pathway by causing the formation of

a receptor I/receptor II tetrameric complex involving two of

each receptor I and receptor II (Figure 1).

The proximity of the receptor I and receptor II serine/

threonine kinase domains induces receptor II to

phosphorylate receptor I, which then phosphorylates the

receptor-regulated Smads (RSmads), Smad2 and Smad3.

Activation of Smad2 and Smad3 cause them to associate

with the Smad4 (co-Smad). The Smad2-Smad3-Smad4

complex then translocates into the nucleus where it binds

other transcription factors, coactivators, and corepressors to

regulate gene expression.

Inhibition of TGFβRI Reduces Levels of Phosphorylated Smads: Elucidation of TGFβ Signaling Using a Multianalyte Immunoassay and Chemical GeneticsRandy Johnston1,2, Tim Warmke1, Rick Wiese1, David Hayes1, Joseph Hwang1

1EMD Millipore Corporation; 2Department of Biomedical Engineering, Washington University, St. Louis, Missouri

Figure 1. One branch of the TGFβ signaling network. Soluble TGFβ binds to a heterodimeric receptor to ultimately activate nuclear translocation of the Smad complex. “TGFβ” actually represents a superfamily of soluble proteins, enabling the cell to integrate a diversity of extracellular signals.

Nuclear membrane

Transcription

TGFβ RII TGFβ RI

Smad2

Smad3

Plasma membrane

TGFβ

Smad7

Smad2

Smad3

Smad4

Smad4

15

Because TGFβ pathway activity can be regulated by relative

levels of multiple proteins, a complete picture of TGFβ

signaling requires simultaneous quantification of these

pathway proteins.

We have developed a bead-based multiplexed immunoassay,

based on Luminex® xMAP® technology, that enables the

simultaneous detection of multiple TGFβ pathway proteins in

a single well, including phosphorylated Smad2, Smad3, ERK,

and Akt, and total TGFβ Receptor II and Smad4 (Table 1).

Using this TGFβ pathway panel, we analyzed the levels

of phosphorylated and total proteins shown in Table 1 in

response to various concentrations of TGFβ2 stimulation,

Protein Phosphorylation state detected

TGFβ Receptor II Total

Erk Thr185/Tyr187

Smad2 Ser465/Ser467

Akt Ser473

Smad3 Ser423/Ser425

Smad4 Total

Table 1. Proteins simultaneously detected in the MILLIPLEX® map 6-plex TGFβ pathway panel.

Materials and MethodsBead conjugationMicrosphere beads were purchased from Luminex

Corporation. Each set of beads is distinguished by different

ratios of two internal dyes yielding a unique fluorescent

signature to each bead set. Capture antibodies (specific to

the proteins listed in Table 1) were covalently coupled to

the carboxylate-modified microsphere beads. We refer to

the resulting bead combination as the MILLIPLEX® map TGFβ

Signaling Pathway Magnetic Bead 6-Plex Panel (Cat. No.

48-614MAG).

Tissue cultureHepG2 cells were cultured according to ATCC® guidelines in

recommended medium. Cells were grown to approximately

85% confluence, then serum-starved for 4 hours prior to

treatment with stimuli or compounds.

Sample preparationTissue samples or cells were lysed and samples collected

according to MILLIPLEX® map Cell Signaling Buffer and

Detection kit instructions. Frozen tissue samples were

weighed and placed on ice. Samples were homogenized

with 1 mL lysis buffer per 30-50 mg tissue. Tissues were

homogenized using the Omni International General

Laboratory Homogenizer with OmniTip™ Plastic Generator

Probes at setting level 2 for 30 seconds. Samples were then

incubated with gentle rocking at 4 ºC for 15 minutes and

the response in analyte levels to TGFβ2 stimulation over a

16 hour time course, the levels of total and phosphorylated

analytes in human breast cancer tissues, and the effect on

pathway proteins by treatment of cells with a commercially

available TGFβRI inhibitor.

centrifuged (10,000 g for 10 minutes at 4 ºC) to separate

connective tissue, fat, ECM, etc. Supernatants were placed

in new tubes. Protein concentration in each sample was

determined by bicinchoninic acid (BCA) assay of sample

aliquots. After diluting samples to 2 mg/mL in lysis buffer,

they were transferred to 96-well plates in preparation for

assay with the MILLIPLEX® map kit.

Bead-based multiplexed immunoassayThe multiplex assay was performed in a 96-well plate

according to product instructions supplied for the

MILLIPLEX® map panel. The plate was first rinsed with 25 µL

assay buffer. 25 µL of controls and samples and 25 µL beads

were added to each well. Plates were incubated overnight

at 4 ºC. Beads were washed twice with assay buffer, then

incubated 1 hour at RT with biotinylated detection antibody

cocktail. The detection antibody cocktail was replaced with

25 µL streptavidin-phycoerythrin (SAPE) and incubated

for 15 minutes at RT. 25 µL of amplification buffer was

added and incubated another 15 minutes at RT. Finally,

the SAPE/amplification buffer was removed and beads

were resuspended in 150 µL assay buffer. The assay plate

was analyzed using a Luminex 200™ system, a compact

unit consisting of an analyzer, a computer, and software

(Luminex Corporation, Austin, TX).

16

ResultsWe treated HepG2 cells with purified TGFβ2, and

simultaneously measured the levels of the six proteins in

Table 1, using the MILLIPLEX® map TGFβ Signaling Pathway

Panel. TGFβ potently activated both Smad2 and Smad3,

with EC50 values of 8.1 pg/mL and 7.8 pg/mL, respectively

(Figure 2).

To determine the kinetics of Smad activation by TGFβ2,

we quantitated phosphorylated Smad2 and Smad3 at

multiple time points following treatment (Figure 3). We

found that activation was initiated after 30 minutes and

reached a maximum at 1-2 h post treatment. Levels of

phosphorylated Smad2 and Smad3 again decreased after 4

h and returned to baseline by 16 h.

TGFβ signaling has been associated with increased cell

motility, and therefore, the progression, invasion and

metastasis of tumors1. Thus, we hypothesized that levels

of activated TGFβ pathway proteins might be increased in

cancer tissues compared to normal tissues. We obtained

two sets of matched breast cancer and normal tissue

samples, prepared lysates and quantified TGFβ pathway

proteins in Table I using the multiplex assay panel. In both

sets, phospho-Smad2 was elevated in the cancer tissues

(Figure 4).

Figure 2. Activation of Smad2 and Smad3 by TGFβ2. Phosphorylated Smad proteins were simultaneously detected in HepG2 cells treated with a 1:4 serial dilution of TGFβ2 ranging from 0.061 pg/mL to 4 ng/mL for 30 minutes.

0

500

1000

1500

2000

2500

0 min 5 min 15 min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr

TGFβ2 time course

MFI

Smad3

0

2000

4000

6000

8000

0 min 5 min 15 min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr

TGFβ2 time course

MFI

Smad2Figure 3. Time Course: Activation of Smad2 and Smad3 by TGFβ2. Phosphorylated Smad proteins were simultaneously detected in HepG2 cells treated with 0.02 ng/mL TGFβ2 for 0, 5, 15, and 30 minutes and 1, 2, 4, 8, and 16 hours.

Figure 4. Phospho-Smad2 was significantly elevated in breast cancer tissue samples. TGFβ pathway proteins were simultaneously detected in two sets (top, bottom) of human tissue samples (20µg/well). Both sets of human matched breast normal and cancer tissue samples were purchased from Asterand.

0

500

1000

1500

2000

2500

0 min 5 min 15 min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr

TGFβ2 time course

MFI

Smad3

0

2000

4000

6000

8000

0 min 5 min 15 min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr

TGFβ2 time course

MFI

Smad2

Phospho-Smad3

-12 -10 -8 -6 -40

1000

2000

3000

4000

EC50 = 7.8 pg/mL

log [TGFβ] (g/L)

MFI

Phospho-Smad2

-12 -10 -8 -6 -40

5000

10000

15000

EC50 = 8.1 pg/mL

log [TGFβ] (g/L)M

FI

0

1000

2000

3000

4000

MFI

Breast Normal 1Breast Cancer 1

0

1000

2000

3000

4000

MFI

Breast Normal 2

Breast Cancer 2

TGFβ2 pERK pSmad2 pAkt pSmad3 Smad4

TGFβ2 pERK pSmad2 pAkt pSmad3 Smad4

0

1000

2000

3000

4000

MFI

Breast Normal 1Breast Cancer 1

0

1000

2000

3000

4000

MFI

Breast Normal 2

Breast Cancer 2

TGFβ2 pERK pSmad2 pAkt pSmad3 Smad4

TGFβ2 pERK pSmad2 pAkt pSmad3 Smad4

17

Because of its correlations with tumor progression and

metastasis, proteins in the TGFβ pathway have emerged

as therapeutic targets, and TGFβ pathway inhibitors have

been developed as therapeutic candidates and for target

validation. One of these inhibitors, TGFβR1 Kinase Inhibitor

II, is a cell-permeable naphthyridinyl pyrazolo compound

that acts as a potent, selective, reversible, and ATP-

competitive inhibitor. We assessed its efficacy in reducing

levels of phosphorylated Smads by treating HepG2 cells

with varying concentrations of inhibitor and measuring

TGFβ pathway proteins in the resulting lysates (Figure 5).

The inhibitor reduced Smad2 phosphorylation with

an EC50 of 28.8 nM (Figure 5A) and reduced Smad3

phosphorylation with an EC50 of 36.5 nM (Figure 5B). The

inhibitor did not affect the levels of the other pathway

proteins measured in the same samples (Figure 5C).

DiscussionUsing a multianalyte immunoassay panel and chemical

genetics using a specific inhibitor, we have demonstrated

that this approach is an efficient method of identifying

connections between signaling events within pathways

made up of multiple proteins operating in concert.

Especially in pathways that involve co-regulation of

multiple proteins, such as the TGFβ pathway, it is important

to assess changes in protein levels simultaneously, in the

same sample, to obtain an accurate picture of pathway

activity. Multiplex assay panels enable this simultaneous

measurement, and also enable the conservation of precious

samples, such as the matched normal/breast cancer tissues

used in this study.

Using small molecule inhibitors to effect loss of function,

known as chemical genetics, facilitates time-course

experiments, because loss of function occurs at a known

time point, unlike traditional or RNAi-mediated experiments.

Understanding the kinetics of inhibition and its relation to

the kinetics of pathway activation is an important step in

the discovery and development of therapeutics.

0

20

40

60

80

100

120

% S

igna

l

- inhibitor+ inhibitor

TGFβ2I pERK pSmad2 pAkt pSmad3 Smad4

Figure 5. Inhibition of TGFβ RI results in decreased levels of phospho-Smad2 and phospho-Smad3. HepG2 cells were pretreated for 30 minutes with TGFβ RI Kinase Inhibitor II (Cat. No. 616452) and then stimulated with 0.05 ng/mL TGFβ2 for 30 minutes. Phorphorylated TGFβ pathway proteins were detected (represented using arbitrary units). The EC50 for the inhibitor on Smad2 (A) and Smad3 (B) activation was calculated using GraphPad Prism® 5 software.

Description Catalogue No.

MILLIPLEX® map TGFβ Signaling Pathway Magnetic Bead 6-Plex 48-614MAG

TGFβRI Kinase Inhibitor II 616452

Available from www.millipore.com.

RELATED PRODUCTS

-10 -9 -8 -6 -5 -40.0

0.5

1.0

1.5

-7

EC50 = 28.8 nM

log (TGFβR1 inhibitor) (M)

Nor

mal

ized

pSm

ad2

signa

l(a

rbitr

ary

unit)

-10 -9 -8 -6 -5 -4

0.0

0.2

0.4

0.6

-7

EC50 = 36.5 nM

log (TGFβR1 inhibitor) (M)

Nor

mal

ized

pSm

ad3

signa

l(a

rbitr

ary

unit)

A.

B.

C.

References1. Leivonen SK and Kähäri VM. Transforming growth factor-beta

signaling in cancer invasion and metastasis. Int J Cancer. 2007 Nov 15;121(10):2119-24.

18

IntroductionNatural killer (NK) cells are a subset of lymphocytes involved

in innate immunity. Their mechanisms of action include

the direct lysis of non-self target cells through release

of perforin and granzymes, as well as the production of

cytokines that are supplied to other innate immune cells,

such as monocytes and macrophages. Given the important

roles that NK cells play in the immune response, the study of

their characteristics and function represents a major field in

immunology.

Although NK cells are typically present within very

heterogeneous immune cell populations, they can be

identified by characteristic cell surface markers. NK cells

are typically CD3-CD56+ lymphocytes; however, CD3- NK

cell subsets include CD56brightCD16– immunoregulatory

NK cells, which express high levels of cytokines, and the

CD56+CD16+ subset of non-cytokine-expressing but highy

cytotoxic NK cells.

CD3+CD56+ NK-T cells are a subset of T cells involved in

recognition of lipid antigens. NK-T cells, however, possess

some properties of NK cells, including cytokine production

and cytotoxicity.

Identification and Functional Characterization of Natural Killer Cells Using Flow CytometryAlex Ko, Don Weldon, Roberto Renteria, Angelica Olcott, Jason Whalley and Matthew HsuEMD Millipore Corporation

Flow cytometry is a popular technique for studying NK cells

in vitro and in vivo. Simultaneous labeling of lymphocytes

with fluorescent antibodies specific for CD16, CD56 and

CD3, followed by flow cytometry analysis, enables the

identification of NK cells and distinguishes them from NK-T

cells. The combination of these three antibodies enables a

researcher to identify natural killer cells from a variety of

relevant source biomaterials, including human PBMCs and

whole blood samples.

Here, we describe the use of a novel flow cytometry kit to

characterize NK cells in human peripheral blood. In addition,

we also describe the use of flow cytometry to perform a cell

cytotoxicity test to detect NK-mediated killing of human

K562 myeloma cells. By measuring both antibody staining

and autofluorescence, we distinguished unbound target

cells and cells that have been targeted by NK cells. We

monitored cell death by the use of a fixable viability dye,

eFluor660®. This dye is excited by the red (633nm) laser and

is fully compatible with cell processing protocols that require

fixation and permeabilization. Because it has an emission

pattern that is specific to the Red2 channel, its signal does

not interfere with the Green and Yellow channels, a problem

commonly associated with propidium iodide staining. The

resulting multiparameter analysis enables the measurement

of viability of specific cell subpopulations within a

heterogeneous sample.

19

Figure 1. Natural Killer (NK) cells are involved in innate immunity, through lysis of target cells. Cytotoxicity is activated through a combination of a positive signal, mediated by an activation receptor on the target, with the lack of an inhibitory signal, which in this case is the absence of major histocompatibility complex I (MHC I) “self” molecule (A). Activation of this response results in the release of perforin and granzyme particles, which act to puncture target cell membrane, and trigger apoptosis, respectively (B).

Activation Receptor

Inhibitory ReceptorMHC Class I

NK+

Target

Activation Receptor Ligand

Cytokine Production

Granule Release(Cytotoxicity)

NK+

Target

-

Materials and MethodsAll cells were analyzed by flow cytometry, using the FlowCellect™ human NK cell

characterization kit, which can be used to stain NK cells from a variety of tissue types,

including PBMCs and whole blood. Flow cytometry was conducted using a guava easyCyte™

benchtop flow cytometry system and the InCyte™ data acquisition and analysis software

package. At least 100,000 events were acquired per sample.

A.

B.

20

4.7% 12.3%

80.7% 2.4%

11.7% 4.7%

18.8% 64.8%

Figure 2. Identification of lymphocyte subsets from whole blood. Fresh human whole blood was stained with anti-CD3 APC, anti-CD16 FITC, and anti-CD56 PE in accordance with the FlowCellect Human NK cell characterization kit user guide. Lymphoid events were further gated for CD16 and CD56 or CD3 and CD56.

Analysis of NK cell subpopulations in whole bloodFresh, human whole blood (10 µL) was added to each well

of a 96-well plates. 5 µL each of anti-CD3 APC, anti-CD16

FITC, and anti-CD56 PE were added to the blood in each

well. The wells were mixed gently and incubated in the

dark at room temperature (RT) for 20 min. After Fixation

Solution (1:40) was added to each well, red blood cells

were lysed by adding 180 µL of Guava 1x Lysing Solution

to each well. Wells were mixed by pipetting and incubated

at RT in the dark for 30 min. After completion of lysis,

samples were centrifuged at 400 x g for 5 minutes and the

supernatant was removed. Cell pellets were washed with

250 µL of 1x Assay Buffer to remove unbound antibodies

and centrifuged at 400 x g for 5 min. Supernatant

was discarded and wash step repeated. Pellets were

resuspended in 250 µL of 1x Assay Buffer prior to analysis.

Cell cytotoxicity testingNegatively selected human NK cells were stained with

anti-CD56 PE, and then incubated with K562 human

myeloma cells at log phase at an effector:target ratio of 5:1.

Following incubation, PE fluorescence combined with green

autofluorescence enabled identification of unbound K562

cells and K562 cells that were being targeted by NK cells.

The eFluor 660® fixable viability dye was then utilized to

identify live cells in each population.

ResultsWe assessed the ability to quantitate lymphocyte subsets

from fresh, human whole blood (Figure 2). By first applying

an elliptical gate to the side scatter v. forward scatter dot

plot, we identified the lymphoid population. We applied this

gate to two dot plots, anti-CD56-PE v. anti-CD16-FITC and

anti-CD56 PE v. anti-CD3 APC. From the resulting statistics,

we determined that approximately 12% of the population

were CD3-CD56+ NK cells and12% of the cells were CD56+

CD16+ NK cells.

21

NK

K562

162 (8%)

1793 (92%)

16.0% dead 4.4% dead

Viability (Bound K562s) Viability (Unbound K562s)

Figure 3. Cytotoxicity of NK cells shown by incubation with K562 human myeloma cells in a 5:1 Effector:Target (E:T) ratio. Panel A shows the distinct scatter patterns between effector and target cells, which enables one to distinguish between the two cell types. Panel B shows the use of CD56 staining and green autofluorescence to distinguish between effector and target cells, as well as between targets that are bound to NK cells from those that are free. Number of each and their percentages are shown. Panels C and D show the viability staining of bound and unbound K562 populations, respectively, with the percentage of dead events in each case.

Next, we used the NK cell characterization kit to assess the proportion of K562 target cells

that were bound to (and being killed by) effector NK cells. In a 5:1 effector cell:target cell

population, 8% of the K562 cells were bound to NK cells (Figure 3B). 84% of the bound K562

cells were viable (3C, stained with fixable viability dye), while 96% of the unbound K562 cells

were viable (Figure 3D).

A. B.

C. D.

22

ConclusionsNatural Killer cells (CD3-CD16+CD56+) are a major player in

innate immunity, both as direct cytotoxic effectors as well

as a regulators for other innate immunity cell types. We

have shown that, using the FlowCellect™ human NK cell

characterization kit, one can achieve accurate phenotyping

on a variety of sample types, including whole blood samples.

Using the same kit to perform an NK cell cytotoxicity test,

we demonstrate that unbound K562 target cells can be

clearly distinguished from those that have been engaged by

CD56-positive NK cells, and each of these populations can be

further investigated for viability using the eFluor 660® dye.

References1. Cooper, MA et al. The biology of human natural killer-cell

subsets. Trends in Immunology. 2001; Vol.22 No. 11: 633-640.2. Renò, F et al. Assays of Natural Killer (NK) Cell Ligation to

Target Cells. Current Protocols in Cytometry. 1998; 9.10.1-9.10.8.

3. Zamai, L et al. Kinetics of In Vitro Natural Killer Activity Against K562 Cells as Detected by Flow Cytometry. Cytometry. 1998; Vol 32: 280-285.

Description Catalogue No.

FlowCellect™ Human Natural Killer Cell Characterization Kit FCIM025164

easyCyte™ 8HT Flow Cytometry System 0500-4008

Related ProductsHelper T Cell KitsMouse

FlowCellect™ Mouse Th1 Intracellular Cytokine Kit FCIM025123

FlowCellect™ Mouse Th2 Intracellular Cytokine Kit FCIM025124

FlowCellect™ Mouse Th17 Intracellular Cytokine Kit FCIM025125

FlowCellect™ Mouse Th1/Th2 Intracellular Cytokine Kit FCIM025137

FlowCellect™ Mouse Th1/Th17 Intracellular Cytokine Kit FCIM025138

Regulatory T Cell KitsHuman

FlowCellect™ Human FOXP3 Treg Characterization Kit FCIM025118

FlowCellect™ Human CD4/CD9 T Cell Kit FCIM100158

Mouse

FlowCellect™ Mouse FOXP3 Treg Identification Kit FCIM025126

FlowCellect™ Mouse Viable Treg Characterization Kit FCIM025168

Available from www.millipore.com.

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23

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NovaCHOice, EmbryoMax, MILLIPLEX, Upstate and Chemicon are registered trademarks and Scepter, Direct Detect, FlowCellect, guava easyCyte, InCyte, Muse, EMD Millipore and the M mark are trademarks of Merck KGaA, Darmstadt, Germany. Trademarks belonging to third parties are the properties of their respective owners. Lit. No. PR4883EN00 04/12 Printed in U.S.A. LS SBU-12-06162© 2012 EMD Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

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