Chris L Langsdorf, Jolene Bradford, Yu-Zhong Zhang, Flow Cytometry Systems, Life Technologies, Eugene, OR, United States RESULTS The addition of a viability dye to remove dead cells from analysis and a primary antibody conjugate to focus the analysis only on the cells of interest further refines this fluorescence histogram. A. The cell-impermeant nucleic acid stain SYTOX® AADvanced™ Dead Cell Stain was used to remove dead cells from analysis. Antibodies can nonspecifically bind to dead cells, introducing error in results. B. A density plot of forward scatter versus side scatter was used to eliminate debris, so that analysis would be focused on intact cells only. C. Proliferation of CD4+ lymphocytes was far more pronounced in this experiment than CD8+ lymphocytes, so a mouse anti-human CD4 Alexa Fluor® 488 primary antibody conjugate was used to gate on CD4+ cells. D. Resolution of individual generations of proliferating cells can be greatly improved by collecting a very large number of events. Peaks outlined in violet represent several generations of dividing cells, with the parent generation represented by the peak on the right and successive generations by peaks to the left. The overlaid peak outlined in red represents undividing control cells which received no stimulus. Figure 2. Strategies to improve CellTrace™ Violet fluorescence histograms Peak resolution and data precision in proliferation experiments can be improved by collecting data from a large number of events. A. This CellTrace™ Violet fluorescence histogram has poor generational resolution because only twenty thousand events are being analyzed. B. Collecting one million events from the same sample of cells greatly improves resolution and leads to better statistical results. As shown below, this data can be further improved with an appropriate gating strategy. Figure 7. Two-way mixed lymphocyte reaction ABSTRACT The mixed lymphocyte reaction (MLR), in which lymphocytes from two donors are combined and co-cultured, has significantly contributed to our understanding of immune function. Initially, the proliferation of responder cells was evaluated by measuring the uptake of radioactive 3 H- thymidine. As technology improved, this method gave way to a technique measuring the incorporation of 5'-bromo-2'-deoxyuridine. Further advances led to assays for detection of incorporated 5'-ethynyl-2'-deoxyuridine and dye-dilution assays using carboxyfluorescein- diacetate succinimidyl ester (CFSE). Here we evaluate another advance in the measurement of lymphocyte proliferation kinetics using CFSE in combination with immunophenotyping and a novel fluorescent cell proliferation tracing dye. In the current study mononuclear cells were isolated from the peripheral blood of healthy human donors using a density gradient and labeled with CFSE or a novel violet cell proliferation dye. One-way MLRs were performed by inhibiting proliferation of antigen-presenting cells with Mitomycin-C. Two-way MLRs were also performed by co-culturing lymphocytes from allogeneic donors. The phenotype, viability, and proliferative index of stimulator and responder cells were evaluated using multicolor flow cytometry. INTRODUCTION The cell proliferation dyes CellTrace™ Violet and CellTrace™ CFSE readily diffuse into live cells where they covalently bind and become intensely fluorescent, providing long- term cell labeling. These dyes have distinct advantages over previous methods of proliferation analysis. Tritiated thymidine incorporation and tetrazolium salt degradation were once used to analyze proliferation, but neither of these approaches permitted positive identification of the proliferative fraction of cells. BrdU incorporation and detection provides a method to identify proliferating cells, but this approach is hindered by a long workflow and the requirement to fix and perm cells. The stable, bright fluorescent label provided by the CellTrace™ dyes permits simple discrimination of individual generations in live cells without the need for antibody detection. Upon cell division each daughter cell receives half of the fluorescent label from the parent cell, resulting in a halving of fluorescence intensity. This serial dye dilution permits identification of distinct generations of proliferating cells. MATERIALS AND METHODS Human peripheral blood mononuclear cells were isolated from the whole blood of healthy donors using a Ficoll-Paque ® Plus density gradient (GE Healthcare). For the first part of the experiment, cells were stained with 10 μM CellTrace™ Violet and resuspended in OpTmizer™ T-Cell Expansion Buffer (GIBCO) containing L-glutamine, penicillin and streptomycin. Stained cells were stimulated to proliferate with 200 ng mouse anti-human CD3 antibody (clone S4.1) and 100 ng Interleukin-2 (IL-2) per milliliter cells and incubated at 37°C and 5% CO 2 for 7 days. Mouse anti-human CD4 Alexa Fluor® 488 and SYTOX ® AADvanced™ Dead Cell Stain were used to gate on live CD4+ lymphocytes. Approximately one million cells were analyzed on an Attune™ Acoustic Focusing Cytometer and proliferation analysis was performed with ModFit LT™ (Verity Software House). CONCLUSIONS •The cell tracing dyes CellTrace™ Violet and CellTrace™ CFSE provide extremely bright, homogenous staining resulting in very little fluorescence variation between cells in a population, enabling the visualization of distinct generations in a fluorescence histogram. •The combination of these two dyes permits long-term tracking of two populations of proliferating cells in one experiment . •Unlike 3 H-Thymidine, the CellTrace™ dyes permit positive identification of proliferating cell populations. •Stable, bright fluorescent detection of proliferation is achieved without the requirement for antibody detection like BrdU. •Collecting data from large numbers of cells improves data precision and resolution of individual generations in proliferation experiments. Superior sample focusing provided by the Attune™ Acoustic Focusing Cytometer enables analysis of very large populations in minimal time without a reduction in data quality. •Experimental results can be improved by the addition of a viability dye and fluorescently-labeled primary antibodies to limit analysis to live cells in the population of interest. REFERENCES 1. J Cell Biol 101, 610 (1985); 2. J Cell Biol 103, 2649 (1986); 3. J Immunol Methods 171, 131 (1994); 4. J Exp Med 184, 277 (1996); 5. J Immunol Methods 133, 87 (1990); 6. Transplant Proc 24, 2820 (1992); 7. Current Protocols in Cytometry, J. P. Robinson, Ed., (1998) pp 9.11.1-9.11.9. 8. Cytometry Part A, 79A: 95–101 (2011). 9. Current Protocols in Immunology 7.10.1-7.10.24, August 2008. ACKNOWLEDGEMENTS Thanks to J. Liu, K. Gee, G. Buller, D. Bantly, and C. Palaniappan TRADEMARKS/LICENSING For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2011 Life Technologies Corporation. All rights reserved. The trademarks herein are the property of Life Technologies Corporation or their respective owners. The next step in the analysis of lymphocyte proliferation Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com Figure 3. Appropriately gated large event file provides superior peak resolution A. C. B. Isolated PBMCs from two donors were treated with 0.5 mg/mL Mitomycin C for 30 min at 37°C or left untreated. Aliquots of cells from each treatment were labeled with 500 nM CellTrace™ CFSE or 10 μM CellTrace™ Violet, then combined in equal amounts and incubated at 37°C and 5% CO 2 for 7 days. Homogenous fluorescence from cells treated with Mitomycin C indicates that these cells did not proliferate, while untreated cells present a fluorescence intensity pattern indicating that one or multiple cell divisions occurred. A. A. B. First Generation Second Generation Third Generation Fourth Generation Brightness Number of Cells Figure 5. Single-color cell tracing with artificial stimulus Figure 6. One-way Mixed Lymphocyte Reactions PBMCs were isolated and labeled with 500 nM CellTrace™ CFSE or 10 μM CellTrace™ Violet prior to stimulation with mouse anti-human CD3 and il-2. Cells were grown at 37°C and 5% CO 2 for 7 days prior to analysis. Live cells were identified by exclusion of SYTOX® AADvanced™ dead cell stain. Figures A-C. Several generations of cells are visible in a CellTrace™ CFSE fluorescence histogram, while very little signal is observed in a CellTrace™ Violet histogram. A density plot helps illustrate the minimal bleed through. D-F. Very little signal is observed in a CellTrace™ CFSE fluorescence histogram, but several generations of cells are visible in a CellTrace™ Violet histogram. The density plot again illustrates the minimal bleed through. A. C. B. D. Donor 1 arrested Aliquots of isolated PBMCs from two healthy human donors were labeled with 500 nM CellTrace™ CFSE or 10 μM CellTrace™ Violet, then combined in equal amounts and incubated at 37°C and 5% CO 2 for 7 days. Live cells were analyzed based on SYTOX® AADvanced™ Dead Cell Stain exclusion. At least 20,000 events were collected using an Attune™ Acoustic Focusing Cytometer with 405 and 488 nm lasers and indicated emission filters. A. Gated regions indicate the proliferative fraction of cells from each donor. In this figure, donor 1 had a higher degree of proliferation. B. Donor 1 again shows a higher degree of proliferation, with no apparent inhibitory effects from either tracing dye. A. B. Contact : [email protected] Proliferating Cells A. C. B. D. F. E. Very little bleed through Donor 2 19.6% proliferating Donor 1 29.9% proliferating Donor 1 32.0% proliferating Donor 2 27% proliferating Donor 2 normal 25.7% proliferating Donor 1 arrested Donor 2 normal 13.4% proliferating Donor 2 arrested Donor 1 normal 13.4% proliferating Donor 2 arrested Donor 1 normal 41.1% proliferating Figure 1. Cell proliferation measurement by dye dilution
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Chris L Langsdorf, Jolene Bradford, Yu-Zhong Zhang, Flow Cytometry Systems, Life Technologies, Eugene, OR, United States
RESULTS
The addition of a viability dye to remove dead cellsfrom analysis and a primary antibody conjugate tofocus the analysis only on the cells of interestfurther refines this fluorescence histogram. A. Thecell-impermeant nucleic acid stain SYTOX®AADvanced™ Dead Cell Stain was used toremove dead cells from analysis. Antibodies cannonspecifically bind to dead cells, introducing errorin results. B. A density plot of forward scatterversus side scatter was used to eliminate debris,so that analysis would be focused on intact cellsonly. C. Proliferation of CD4+ lymphocytes was farmore pronounced in this experiment than CD8+lymphocytes, so a mouse anti-human CD4 AlexaFluor® 488 primary antibody conjugate was usedto gate on CD4+ cells. D. Resolution of individualgenerations of proliferating cells can be greatlyimproved by collecting a very large number ofevents. Peaks outlined in violet represent severalgenerations of dividing cells, with the parentgeneration represented by the peak on the rightand successive generations by peaks to the left.The overlaid peak outlined in red representsundividing control cells which received no stimulus.
Figure 2. Strategies to improve CellTrace™ Violet fluorescence histograms
Peak resolution and data precision in proliferation experiments can be improved by collecting datafrom a large number of events. A. This CellTrace™ Violet fluorescence histogram has poorgenerational resolution because only twenty thousand events are being analyzed. B. Collecting onemillion events from the same sample of cells greatly improves resolution and leads to betterstatistical results. As shown below, this data can be further improved with an appropriate gatingstrategy.
Figure 7. Two-way mixed lymphocyte reactionABSTRACT
The mixed lymphocyte reaction (MLR), in which lymphocytes from two donors are combined andco-cultured, has significantly contributed to our understanding of immune function. Initially, theproliferation of responder cells was evaluated by measuring the uptake of radioactive 3H-thymidine. As technology improved, this method gave way to a technique measuring theincorporation of 5'-bromo-2'-deoxyuridine. Further advances led to assays for detection ofincorporated 5'-ethynyl-2'-deoxyuridine and dye-dilution assays using carboxyfluorescein-diacetate succinimidyl ester (CFSE). Here we evaluate another advance in the measurement oflymphocyte proliferation kinetics using CFSE in combination with immunophenotyping and anovel fluorescent cell proliferation tracing dye. In the current study mononuclear cells wereisolated from the peripheral blood of healthy human donors using a density gradient and labeledwith CFSE or a novel violet cell proliferation dye. One-way MLRs were performed by inhibitingproliferation of antigen-presenting cells with Mitomycin-C. Two-way MLRs were also performedby co-culturing lymphocytes from allogeneic donors. The phenotype, viability, and proliferativeindex of stimulator and responder cells were evaluated using multicolor flow cytometry.
INTRODUCTION
The cell proliferation dyes CellTrace™ Violet and CellTrace™ CFSE readily diffuse intolive cells where they covalently bind and become intensely fluorescent, providing long-term cell labeling. These dyes have distinct advantages over previous methods ofproliferation analysis. Tritiated thymidine incorporation and tetrazolium salt degradationwere once used to analyze proliferation, but neither of these approaches permittedpositive identification of the proliferative fraction of cells. BrdU incorporation anddetection provides a method to identify proliferating cells, but this approach is hinderedby a long workflow and the requirement to fix and perm cells. The stable, brightfluorescent label provided by the CellTrace™ dyes permits simple discrimination ofindividual generations in live cells without the need for antibody detection. Upon celldivision each daughter cell receives half of the fluorescent label from the parent cell,resulting in a halving of fluorescence intensity. This serial dye dilution permitsidentification of distinct generations of proliferating cells.
MATERIALS AND METHODS
Human peripheral blood mononuclear cells were isolated from the whole blood of healthy donorsusing a Ficoll-Paque® Plus density gradient (GE Healthcare). For the first part of the experiment,cells were stained with 10 µM CellTrace™ Violet and resuspended in OpTmizer™ T-CellExpansion Buffer (GIBCO) containing L-glutamine, penicillin and streptomycin. Stained cellswere stimulated to proliferate with 200 ng mouse anti-human CD3 antibody (clone S4.1) and 100ng Interleukin-2 (IL-2) per milliliter cells and incubated at 37°C and 5% CO2 for 7 days. Mouseanti-human CD4 Alexa Fluor® 488 and SYTOX® AADvanced™ Dead Cell Stain were used togate on live CD4+ lymphocytes. Approximately one million cells were analyzed on an Attune™Acoustic Focusing Cytometer and proliferation analysis was performed with ModFit LT™ (VeritySoftware House).
CONCLUSIONS
•The cell tracing dyes CellTrace™ Violet and CellTrace™ CFSE provide extremely bright, homogenous staining resulting in very little fluorescence variation between cells in a population, enabling the visualization of distinct generations in a fluorescence histogram.•The combination of these two dyes permits long-term tracking of two populations of proliferating cells in one experiment .•Unlike 3H-Thymidine, the CellTrace™ dyes permit positive identification of proliferating cell populations. •Stable, bright fluorescent detection of proliferation is achieved without the requirement for antibody detection like BrdU.•Collecting data from large numbers of cells improves data precision and resolution of individual generations in proliferation experiments. Superior sample focusing provided by the Attune™ Acoustic Focusing Cytometer enables analysis of very large populations in minimal time without a reduction in data quality. •Experimental results can be improved by the addition of a viability dye and fluorescently-labeled primary antibodies to limit analysis to live cells in the population of interest.
REFERENCES1. J Cell Biol 101, 610 (1985); 2. J Cell Biol 103, 2649 (1986); 3. J Immunol Methods 171, 131 (1994); 4. J Exp Med 184, 277 (1996); 5. J Immunol Methods 133, 87 (1990); 6. Transplant Proc 24, 2820 (1992); 7. Current Protocols in Cytometry, J. P. Robinson, Ed., (1998) pp 9.11.1-9.11.9. 8. Cytometry Part A, 79A: 95–101 (2011). 9. Current Protocols in Immunology 7.10.1-7.10.24, August 2008.
ACKNOWLEDGEMENTSThanks to J. Liu, K. Gee, G. Buller, D. Bantly, and C. Palaniappan
TRADEMARKS/LICENSINGFor Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. ©2011 Life Technologies Corporation. All rights reserved. The trademarks herein are the propertyof Life Technologies Corporation or their respective owners.
The next step in the analysis of lymphocyte proliferation
Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
Figure 3. Appropriately gated large event file provides superior peak resolution A. C.B.
Isolated PBMCs from two donors were treated with 0.5 mg/mL Mitomycin C for 30 min at 37°C or leftuntreated. Aliquots of cells from each treatment were labeled with 500 nM CellTrace™ CFSE or 10µM CellTrace™ Violet, then combined in equal amounts and incubated at 37°C and 5% CO2 for 7days. Homogenous fluorescence from cells treated with Mitomycin C indicates that these cells did notproliferate, while untreated cells present a fluorescence intensity pattern indicating that one or multiplecell divisions occurred. A.
A. B.
First Generation
Second Generation
Third Generation
Fourth Generation
Brightness
Num
ber o
f Cel
ls
Figure 5. Single-color cell tracing with artificial stimulus
Figure 6. One-way Mixed Lymphocyte Reactions
PBMCs were isolated and labeled with 500 nM CellTrace™ CFSE or 10 µM CellTrace™ Violet prior tostimulation with mouse anti-human CD3 and il-2. Cells were grown at 37°C and 5% CO2 for 7 days priorto analysis. Live cells were identified by exclusion of SYTOX® AADvanced™ dead cell stain. FiguresA-C. Several generations of cells are visible in a CellTrace™ CFSE fluorescence histogram, while verylittle signal is observed in a CellTrace™ Violet histogram. A density plot helps illustrate the minimalbleed through. D-F. Very little signal is observed in a CellTrace™ CFSE fluorescence histogram, butseveral generations of cells are visible in a CellTrace™ Violet histogram. The density plot againillustrates the minimal bleed through.
A.
C.
B.
D.
Donor 1 arrested
Aliquots of isolated PBMCs from two healthy human donors were labeled with 500 nM CellTrace™CFSE or 10 µM CellTrace™ Violet, then combined in equal amounts and incubated at 37°C and 5%CO2 for 7 days. Live cells were analyzed based on SYTOX® AADvanced™ Dead Cell Stain exclusion.At least 20,000 events were collected using an Attune™ Acoustic Focusing Cytometer with 405 and488 nm lasers and indicated emission filters. A. Gated regions indicate the proliferative fraction of cellsfrom each donor. In this figure, donor 1 had a higher degree of proliferation. B. Donor 1 again shows ahigher degree of proliferation, with no apparent inhibitory effects from either tracing dye.
A. B.
Contact : [email protected]
Proliferating Cells
A. C.B.
D. F.E.
Very little bleed through
Donor 219.6% proliferating
Donor 129.9% proliferating
Donor 132.0% proliferating
Donor 227% proliferating
Donor 2 normal 25.7% proliferating
Donor 1 arrested
Donor 2 normal 13.4% proliferating
Donor 2 arrested
Donor 1 normal 13.4% proliferating
Donor 2 arrested
Donor 1 normal 41.1% proliferating
Figure 1. Cell proliferation measurement by dye dilution
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