Measurement of Proliferative Responses of Cultured Lymphocytes

Linda Mesler Muul1, Guido Heine2, Christopher Silvin1, Stephen P. James3, Fabio Candotti1, Andreas Radbruch2, Margitta Worm2

1 GMBB, National Human Genome Institute, Bethesda, Maryland, 2 Deutsches Rheuma‐Forschungszentrum Berlin, Berlin, Germany, 3 University of Maryland School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.10
DOI:  10.1002/0471142735.im0710s94
Online Posting Date:  August, 2011
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Abstract

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to induce proliferation in lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying protocol, which contains methods to determine cell proliferation by incorporation of [3H]thymidine into DNA by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT or WST‐1). These protocols provide an estimate of cell proliferation indirectly by measuring DNA synthesis, and cell metabolic activity in an entire cell population, but no data on individual cells is obtained. A protocol for CFSE labeling allows direct detection of single proliferating cells and facilitates the quantification of cell divisions by flow cytometry according to the respective CFSE‐dilution, and following costaining with fluorescent labeled antibodies, the characterization of subpopulations in the cell culture. Curr. Protoc. Immunol. 94:7.10.1‐7.10.26. © 2011 by John Wiley & Sons, Inc.

Keywords: lymphocytes; proliferation; mitogens/antigens; CFSE; mixed lymphocyte reaction (MLR); WST‐1

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture for Mitogen‐Induced Proliferation of Peripheral Blood Mononuclear Cells
  • Alternate Protocol 1: Culture for One‐Way Mixed Lymphocyte Reaction
  • Alternate Protocol 2: Culture for Autologous Mixed Lymphocyte Reaction
  • Alternate Protocol 3: Culture for Antigen‐Induced T Cell Proliferation
  • Alternate Protocol 4: Culture for Lymphokine‐Dependent B Cell Proliferation
  • Basic Protocol 2: Assay of PBMC Response to Mitogens, Antigens, and Lymphokines
  • Basic Protocol 3: Measuring Proliferation of Human Lymphocytes Using Succinimidyl Ester of Carboxyfluorescein Diacetate (CFSE)
  • Support Protocol 1: Preparation of Platelet‐Poor, Heat‐Inactivated Human Plasma
  • Support Protocol 2: [3H]Thymidine Pulse and Harvest of Cell Cultures
  • Support Protocol 3: Quantitative Assessment of Cell Proliferation Using MTT Tetrazolium Salt
  • Support Protocol 4: Quantitative Assessment of Cell Proliferation Using WST‐1 Tetrazolium Saltx
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culture for Mitogen‐Induced Proliferation of Peripheral Blood Mononuclear Cells

  Materials
  • Peripheral blood mononuclear cells (PBMC) suspension (unit 7.1) in medium to be used for the assay
  • Complete RPMI medium ( appendix 2A) containing 5% to 10% human AB serum (heat‐inactivated 30 to 60 min at 56°C) or platelet‐poor plasma ( protocol 8) or, alternatively, a commercially available serum‐free medium designed for human lymphocytes (e.g., X‐Vivo, Lonza; AIM V, Invitrogen)
  • 100 µg/ml phytohemagglutinin (PHA; see Table 7.10.1) in culture medium used in the assay (store in aliquots up to 2 years at –20°C)
  • 96‐well flat‐bottom (recommended for tetrazolium assays) or round‐bottom microtiter plates, with lids
  • Humidified 37°C, 5% CO 2 cell culture incubator
  • Additional reagents and equipment for counting cells ( appendix 3A)
    Table 7.0.1   MaterialsCommon Human Lymphocyte Activators

    Cell type a Concentrations b Supplier c
    Activator antigens
    Tetanus toxoid T cells, B cells 0.1‐12 LF/ml 0.1‐20 µg/ml Statens Serum Institut d, Calbiochem, CSL e, Novartis (Chiron Behring), RIVM f
    Candida albicans T cells, B cells 10‐20 µg/ml Greer Labs, Artu Biologicals g, Hollister Stier h
    CMV T cells, B cells 2 µM, 1:20 dilution Beckman Coulter, EastCoast Bio j, UCHSC Pediatric Infectious Diseases k
    Rubeola T cells, B cells 0.75 µg/ml Viral Antigens l, EastCoast Bio j
    EBV T cells, B cells 2 µM Beckman Coulter, EastCoast Bio j
    HSV T cells, B cells 1:20 dilution UCHSC Pediatric Infectious Diseases k, EastCoast Bio j
    HIV T cells, B cells 1‐10 µg/ml Protein Sciences m, Austral Biologicals n, EastCoast Bio j
    VZV T cells, B cells 1:20 dilution UCHSC Pediatric Infectious Diseases k, EastCoast Bio j
    Streptolysin O T cells, B cells 5 µg/ml Sigma
    Staphylococcus aureus Cowan B cells 50 µg/ml Calbiochem
    Superantigens
    SEA T cells 10 ng‐1 µg/ml Sigma, Toxin Technology
    SEB T cells 10 ng‐1 µg/ml Sigma, Toxin Technology
    SEC T cells 10 ng‐1 µg/ml Toxin Technology
    SED T cells 10 ng‐1 µg/ml Toxin Technology
    SEE T cells 10 ng‐1 µg/ml Toxin Technology
    Mitogens
    PHA T cells 1‐10 µg/ml Sigma, Roche, Invitrogen, EY Laboratories , MP Biomedical
    ConA T cells 1‐50 µg/ml Sigma, EY Laboratories
    PWM T cells, B cells 1‐10 µg/ml Sigma, EY Laboratories
    TCR antibodies
    Anti‐CD3 T cells 50 ng‐1 µg/ml Ortho Biotech , BD, ATCC, Abcam
    Anti‐CD3/CD28 T cells 50 ng‐1 µg/ml Ortho Biotech , PharMingen
    Anti‐CD3/CD28 beads T cells 1:20‐1:200, three beads/cell Invitrogen
    Anti‐CD3, CD80 (B7.1 Ig) T cells 100 ng/ml, 4 µg/ml Ortho Biotech , R&D Systems
    Anti‐CD3, Anti‐CD6 T cells 1‐100 ng/ml, 100 ng/ml Ortho Biotech , Dako, Abcam
    Anti‐CD3, Anti‐LFA T cells 1 µg/ml, 10 µg/ml Ortho Biotech , ATCC, Abcam
    Anti‐CD3, anti‐ICAM‐1 T cells 1 µg/ml, 10 µg/ml Ortho Biotech , ATCC, Abcam
    Anti‐CD3, rVCAM‐1 T cells 10 µg/ml, 10 µg/ml Ortho Biotech , Genentech
    Anti‐CD3, fibronectin T cells 1 µg/ml, 50 ng/ml Ortho Biotech , BD, ATCC, Abcam , Invitrogen
    Phorbol ester
    PMA T cells 0.5‐10 ng/ml Sigma
    PMA/ionomycin T cells 0.5‐10 ng/ml, 0.5 µM Sigma, Calbiochem
    Cytokine/lymphokines
    IL‐2 T cells 1‐3000 U/ml Roche, R&D Systems, Genzyme, PeproTech
    IL‐4 T cells 1‐1000 U/ml Genzyme, R&D Systems, Promega, PeproTech
    IL‐7 + IL‐15 + TNFa + IL‐6 T cells 25 ng/ml each BD, R&D Systems, Roche, PeproTech
    IL‐2 + IL‐12 T cells 5‐25 U/ml, 125 U/ml R&D Systems, PeproTech
    Chemokines
    Anti‐CD3 + CCL5 T cells 1 µg/ml, 10 nM Ortho Biotech , PeproTech
    Anti‐CD3 + CXCL12 T cells 1 µg/ml, 10 nM Ortho Biotech , PeproTech
    Other
    Anti‐IgM B cells 5‐50 µg/ml Bio‐Rad, Jackson ImmunoResearch Laboratories, Abcam
    Anti‐IgM + anti‐CD40 B cells 10‐50 µg/ml, 100 ng/ml Bio‐Rad, ATCC, Abcam
    Anti‐IgM + IL‐4 B cells 10‐50 µg/ml, 50 U/ml Bio‐Rad, Abcam , Schering Plough, R&D Systems
    Anti‐CD40 B cells 100 ng‐2.5 µg/ml ATCC, Abcam , R&D Systems
    Anti‐CD40 + IL‐1 B cells 100 ng/ml, 2.5‐100 U/ml ATCC, Abcam, Genzyme, R&D Systems, Roche, PeproTech
    Anti‐CD40 + IL‐2 B cells 100 ng/ml, 1‐50 U/ml ATCC, Abcam , Genzyme, R&D Systems, Roche, PeproTech
    Anti‐CD40 + IL‐3 B cells 100 ng/ml, 1‐50 ng/ml ATCC, Abcam, Schering Plough, R&D Systems, PeproTech
    Anti‐CD40 + IL‐4 B cells 100 ng/ml, 1‐1,750 U/ml ATCC, Abcam , Genzyme, Schering Plough, PeproTech
    Anti‐CD40 + IL‐6 B cells 100 ng/ml, 0.2‐25 ng/ml ATCC, Abcam , Genzyme, R&D Systems, Roche, PeproTech
    Anti‐CD40 + IL‐7 B cells 100 ng/ml, 1‐100 U/ml ATCC, Abcam , Schering Plough, R&D Systems, PeproTech
    Anti‐CD40 + IL‐2 + IL‐10 B cells 0.5 µg/ml, 100 U/ml, 100 ng/ml ATCC, Abcam , R&D Systems, PeproTech
    Anti‐CD40 + IL‐21 B cells 1 µg/ml, 100 ng/ml ATCC, Abcam , ZymoGenetics , Invitrogen
    CpG + IL‐2 B cells 1 µg/ml, 10‐100 U/ml Oligos etc, MWG Biotech, Genzyme, R&D Systems, Roche, BD, PeproTech
    CpG + IL‐15 B cells 1 µg/ml, 5‐10 ng/ml Oligos etc, R&D Systems, PeproTech
    Staphylococcus aureus Cowan + IL‐2 B cells 0.01%, 10‐100 U/ml Calbiochem, Genzyme, R&D Systems, Roche, BD
    Activated mitomycin C–treated T cells B cells Sigma

     aCell types that respond to the stimulus.
     bConcentrations given are ranges. Depending on the system, lower or higher concentrations may be better. All reagents should be titrated for particular applications. Since human response is variable, multiple donors and multiple doses should be tested.
     cSuppliers listed are the most common, but are not necessarily the only ones, nor may they be considered the recommended ones, but only a place to start. See appendix 55 or designated footnotes for information about suppliers.
     dhttp://www.ssi.dk/sw379.asp
     ehttp://www.csl.gov.uk/index.cfm
     fhttp://rivm.openrepository.com/rivm
     ghttp://www.artu‐biologicals.nl
     hhttp://www.hollisterstier.com
     Jhttp://www.eastcoastbio.com
     khttp://www.uchsc.edu/peds/subs/id/index.htm
     lhttp://meridianlifescience.com/about_us/about_vai.asp
     mhttp://www.proteinsciences.com
     nhttp://www.australbiologicals.com
     http://www.toxintechnology.com
     http://www.eylabs.com
     http://www.mpbio.com
     http://www.orthobiotech.com
     http://www.abcam.com
     http://www.peprotech.com
     http://www.zymogenetics.com
     Abbreviation: LF, limits of flocculation.

Alternate Protocol 1: Culture for One‐Way Mixed Lymphocyte Reaction

  • Autologous cell suspension (PBMC; unit 7.1)
  • Allogeneic cell suspension (PBMC; unit 7.1)
  • Normal donor cell suspension (PBMC; unit 7.1)
  • Cell irradiator or 0.5 mg/ml mitomycin C (Sigma) solution in complete RPMI medium ( appendix 2A) containing 5% to 10% (v/v) autologous human serum or platelet‐poor plasma ( protocol 8): store up to 1 year at –80°C, protected from light; discard if precipitate present

Alternate Protocol 2: Culture for Autologous Mixed Lymphocyte Reaction

  • T cells from autologous individual (unit 7.2) or peripheral blood mononuclear cells (PBMC) from autologous individual (unit 7.1)
  • Non‐T cells (unit 7.2 or unit 7.6) or PBMC (unit 7.1) from autologous individual
  • Normal donor PBMC (unit 7.1)
  • Cell irradiator or 0.5 mg/ml mitomycin C (Sigma) solution in complete RPMI medium ( appendix 2A) containing 5% to 10% (v/v) autologous human serum or platelet‐poor plasma ( protocol 8): store up to 1 year at –80°C, protected from light; discard if precipitate is present

Alternate Protocol 3: Culture for Antigen‐Induced T Cell Proliferation

  • T cell suspension (units 7.2& 7.4)
  • Autologous antigen‐presenting cell suspension: non‐T cells (unit 7.2) or autologous monocytes/macrophages (unit 7.6)
  • PBMC from two normal donors (control; two to ensure at least one positive in vitro response; not all normal individuals respond to the antigen)
  • Antigens to be tested, e.g., tetanus toxoid: prepared in a series of dilutions that will allow 50 µl to give the appropriate final concentration (0, 5, 10, and 20 µg/ml) in each 200‐µl well
  • Cell irradiator or 0.5 mg/ml mitomycin C (Sigma) solution in complete RPMI medium ( appendix 2A) containing 5% to 10% (v/v) autologous human serum or platelet‐poor plasma ( protocol 8): store up to 1 year at –80°C, protected from light; discard if precipitate is present

Alternate Protocol 4: Culture for Lymphokine‐Dependent B Cell Proliferation

  • Tonsil B cell suspension (units 7.5& 7.8) or purified B cells from PBMC (unit 7.5)
  • 5 µg/ml anti‐human CD 40 (see Table 7.10.1) in complete RPMI or alternative medium ( protocol 1)
  • 25 µg/ml anti‐human IgM, azide free (see Table 7.10.1) in complete RPMI or alternative medium ( protocol 1)
  • 10,000 U/ml human rIL‐4 (see Table 7.10.1) in complete RPMI or alternative medium ( protocol 1)

Basic Protocol 2: Assay of PBMC Response to Mitogens, Antigens, and Lymphokines

  Materials
  • Peripheral blood mononuclear cells (PBMC) suspension from test subject or patient (unit 7.1)
  • PBMC suspension from healthy individuals (more than one) as controls (unit 7.1)
  • Complete RPMI medium ( appendix 2A) containing 5% to 10% human AB serum (heat‐inactivated 30 to 60 min at 56°C) or platelet‐poor, heat‐inactivated human plasma ( protocol 8) or a commercially available serum‐free medium designed for human lymphocytes
  • Antigens and mitogens (see Table 7.10.1), e.g., anti‐CD3, phytohemagglutinin (PHA), IL‐2, concanavalin A (con A), diphtheria toxoid, tetanus toxoid (TT), pokeweed mitogen (PWM), Candida albicans (see Table 7.10.1)
  • Flat‐bottom 96‐well microtiter plates
  • Additional reagents and equipment for counting cells ( appendix 3A)

Basic Protocol 3: Measuring Proliferation of Human Lymphocytes Using Succinimidyl Ester of Carboxyfluorescein Diacetate (CFSE)

  Materials
  • Peripheral blood mononuclear cells (PBMC; unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4, containing 0.1% (v/v) human serum albumin (HSA; Gemini BioProducts), sterile
  • CFSE stock suspension (5 mM; see recipe)
  • Complete RPMI medium (see appendix 2A) with 10% (v/v) HSA (Gemini BioProducts), cold
  • Complete RPMI medium ( appendix 2A) containing 5% to 10% (v/v) human AB serum (heat‐inactivated 30 to 60 min at 56°C) or platelet‐poor, heat‐inactivated human plasma ( protocol 8)
  • Appropriate stimuli (e.g., PHA, PWM, ConA, TT, C. albicans)
  • Wash buffer: PBS, pH 7.4 ( appendix 2A), containing 2% (v/v) human serum or plasma ( protocol 8)
  • Cell surface–staining antibodies: e.g., PerCP‐labeled CD3 (e.g., BD Biosciences), PE‐labeled CD8 (e.g., BD Biosciences), and APC‐labeled CD4 (e.g., BD Biosciences), 1 mM propidium iodide (Invitrogen, Sigma)
  • Sterile transfer pipets
  • 37°C incubator
  • Light‐proof tissue
  • Round‐bottom 96‐well microtiter plates
  • 12 × 75–mm FACS tubes
  • Flow cytometer (FACS; see Commentary), capable of analyzing three or four colors
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 1: Preparation of Platelet‐Poor, Heat‐Inactivated Human Plasma

  Materials
  • Plasma from six to ten human volunteers with type A+ blood
  • 500‐ or 100‐ml pipets, sterile
  • 250‐ or 500‐ml conical centrifuge tubes
  • Centrifuge
  • 56°C water bath
  • 50‐ml tubes
IMPORTANT NOTE: Be sure to keep everything sterile to obviate the need to filter sterilize the human plasma.

Support Protocol 2: [3H]Thymidine Pulse and Harvest of Cell Cultures

  Materials
  • Experimental cell cultures in 96‐well microtiter plates (Basic Protocol protocol 11 or protocol 62 or Alternate Protocols protocol 21, protocol 32, protocol 43, or protocol 54)
  • 50 µCi/ml 3H‐labeled methyl thymidine, sterile aqueous formulation (6.7 Ci/mM; Amersham or ICN Biomedicals) in complete medium labeled in the 5 methyl position (recommended) to avoid labeling RNA by conversion of thymidine into uridine
  • Scintillation fluid for nonaqueous 3H‐labeled samples (e.g., Ready Safe, Beckman)
  • Multiwell harvester, preferably semiautomated (e.g., Cambridge Technology, Beckman, Pharmacia LKB Biotechnology, or Skatron)
  • Filter paper for cell harvester
  • Forceps
  • Scintillation counter and vials
CAUTION: Use appropriate procedures and precautions for handling radioactive materials. Special shielding is not required, as [3H] emits low energy beta particles. See appendix 1Q.

Support Protocol 3: Quantitative Assessment of Cell Proliferation Using MTT Tetrazolium Salt

  Materials
  • 5 mg/ml 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazoliumbromide (MTT) (e.g., Sigma, Calbiochem) stock solution: prepare in phosphate‐buffered saline (PBS; appendix 2A) and filter to remove particles; store in aliquots up to 6 months at –20°C
  • Experimental cell cultures in 96‐well microtiter plates (Basic Protocol protocol 11 or protocol 62 or Alternate Protocols protocol 21, protocol 32, protocol 43, or protocol 54)
  • Solubilizing solution (see recipe)
  • Multichannel or repeating pipettor
  • 37°C incubator
  • Centrifuge with rotor adapted for 96‐well plates
  • Microplate spectrophotometer with 570‐nm filter (e.g., Thermo Scientific, BioTek)
CAUTION: Wear gloves to handle MTT, as it is a potential carcinogen.

Support Protocol 4: Quantitative Assessment of Cell Proliferation Using WST‐1 Tetrazolium Saltx

  Materials
  • Experimental cell cultures in 96‐well microtiter plates (Basic Protocol protocol 11 or protocol 62 or Alternate Protocols protocol 21, protocol 32, protocol 43, or protocol 54)
  • Cell culture medium
  • Test reagents
  • WST‐1 reagent (e.g., Roche, Germany)
  • 37°C, 5% CO 2 humidified incubator
  • Microplate spectrophotometer with 450‐ and 680‐nm filter (e.g., MWG, Thermo Scientific, BioTek)
  • Additional reagents and equipment for counting cells ( appendix 3A)
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Literature Cited

Literature Cited
   Alouf, J.E. and Muller‐Alouf, H. 2003. Staphylococcal and streptococcal superantigens: Molecular, biological and clinical aspects. Int. J. Med. Microbiol. 292:429‐440.
   Angulo, R. and Fulcher, D.A. 1998. Measurement of Candida‐specific blastogenesis: Comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression. Cytometry 34:143‐151.
   Berridge, M.V., Herst, P.M., and Tan, A.S. 2005. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnol. Annu. Rev. 11:127‐152.
   Boothby, M., Mora, A.L., and Stephenson, L.M. 2001. Lymphokine‐dependent proliferation of T‐lymphoid cells: Regulated responsiveness and role in vivo. Crit. Rev. Immunol. 21:487‐522.
   Callard, R. and Hodgkin, P. 2007. Modeling T‐ and B cell growth and differentiation. Immunol. Rev. 216:119‐129.
   Engelke, M., Engels, N., Dittmann, K., Stork, B., and Wienands, J. 2007. Ca(2+) signaling in antigen receptor‐activated B lymphocytes. Immunol. Rev. 218:235‐246.
   Geginat, J., Sallusto, F., and Lanzavecchia, A. 2003. Cytokine‐driven proliferation and differentiation of human naive, central memory and effector memory CD4+ T cells. Pathol. Biol. 51:64‐66.
   Gieni, R.S., Li, Y., and HayGlass, K.T. 1995. Comparison of [3H]thymidine incorporation with MTT‐ and MTS‐based bioassays for human and murine IL‐2 and IL‐4 analysis. Tetrazolium assays provide markedly enhanced sensitivity. J. Immunol. Methods 187:85‐93.
   Godfrey, W.R., Krampf, M.R., Taylor, P.A., and Blazar, B.R. 2004. Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation. Blood 103:1158‐1165.
   Hearing, S.D., Norman, M., and Dayan, C.M. 1999. In vitro measurement of lymphocyte steroid sensitivity: Lack of agreement between whole blood culture and separated lymphocyte culture. Immunopharmacol. Immunotoxicol. 21:41‐53.
   Heine, G., Dahten, A., Hilt, K., Ernst, D., Milovanovic, M., Hartmann, B., and Worm, M. 2009. Liver X receptors control IgE expression in B cells. J. Immunol. 182:5276‐5282.
   Hodgkin, P.D., Hawkins, E.D., Hasbold, J., Gett, A.V., Deenick, E.K., Todd, E.F., andHommel, M. 2005. Monitoring T cell proliferation. In Analyzing T Cell Responses ( D. Nagorsen and F.M. Marincola, eds.) pp. 123‐142. Springer, Dordrecht, The Netherlands.
   Hoffmeister, B., Kiecker, F., Tesfa, L., Volk, H.D., Picker, L.J., and Kern, F. 2003. Mapping T cell epitopes by flow cytometry. Methods 29:270‐281.
   Koelle, D.M. 2003. Expression cloning for the discovery of viral antigens and epitopes recognized by T cells. Methods 29:213‐226.
   Kracker, S. and Radbruch, A. 2004. Immunoglobulin class switching: In vitro induction and analysis. Methods Mol. Biol. 271:149‐159.
   Krakauer, T. 1999. Immune response to staphylococcal superantigens. Immunol. Res. 20:163‐173.
   Mannering, S.I., Dromey, J.A., Morris, J.S., Thearle, D.J., Jensen, K.P., and Harrison, L.C. 2005. An efficient method for cloning human autoantigen‐specific T cells. J. Immunol. Meth. 298:83‐92.
   Marin, L., Minguela, A., Torio, A., Moya‐Quiles, M.R., Muro, M., Montes‐Ares, O., Parrado, A., Alvarez‐Lopez, D.M., and Garcia‐Alonso, A.M. 2003. Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture. Cytometry A 51:107‐118.
   Marshall, N.J., Goodwin, C.J., and Holt, S.J. 1995. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regul. 5:69‐84.
   Mueller, D.L. 2000. T cells: A proliferation of costimulatory molecules. Curr. Biol. 10:R227‐R230.
   Niks, M., Otto, M., Busova, B., and Stefanovic, J. 1990. Quantification of proliferative and suppressive responses of human T lymphocytes following ConA stimulation. J. Immunol. Methods 126:263‐271.
   O'Nions, J. and Allday, M.J. 2004. Proliferation and differentiation in isogenic populations of peripheral B cells activated by Epstein‐Barr virus or T cell‐derived mitogens. J. Gen. Virol. 85:881‐895.
   Parish, C.R. 1999. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77:499‐508.
   Piekoszewski, W., Chow, F.S., and Jusko, W.J. 1994. Inhibition of phytohaemagglutinin‐induced lymphocyte proliferation by immunosuppressive drugs: Use of whole blood culture. Immunopharmacol. Immunotoxicol. 16:389‐401.
   Rasooly, L., Rose, N.R., Shah, D.B., and Rasooly, A. 1997. In vitro assay of Staphylococcus aureus enterotoxin A activity in food. Appl. Environ. Microbiol. 63:2361‐2365.
   Scheinberg, P., Price, D.A., Ambrozak, D.R., Barrett, A.J., and Douek, D.C. 2006. Alloreactive T cell clonotype recruitment in a mixed lymphocyte reaction: Implications for graft engineering. Exp. Hematol. 34:788‐795.
   Schluns, K.S. and Lefrancois, L. 2003. Cytokine control of memory T cell development and survival. Nat. Rev. 3:269‐279.
   Suni, M.A., Maino, V.C., and Maecker, H.T. 2005. Ex vivo analysis of T cell function. Curr. Opin. Immunol. 17:434‐440.
   Tangye, S.G. and Hodgkin, P.D. 2004. Divide and conquer: The importance of cell division in regulating B cell responses. Immunology 112:509‐520.
   Tasker, L. and Marshall‐Clarke, S. 2003. Functional responses of human neonatal B lymphocytes to antigen receptor cross‐linking and CpG DNA. Clin. Exp. Immunol. 134:409‐419.
   Thiel, A., Scheffold, A., and Radbruch, A. 2004. Antigen‐specific cytometry—new tools arrived! Clin. Immunol. 111:155‐161.
   Van Kooten, C., Galibert, L., Seon, B.K., Garrone, P., Liu, Y.J., and Banchereau, J. 1997. Cross‐linking of antigen receptor via Ig‐beta (B29, CD79b) can induce both positive and negative signals in CD40‐activated human B cells. Clin. Exp. Immunol. 110:509‐515.
   Venken, K., Thewissen, M., Hellings, N., Somers, V., Hensen, K., Rummens, J.L., and Stinissen, P. 2007. A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto‐antigen specific and polyclonal T cell responses. J. Immunol. Methods 322:1‐11.
   Weichert, H., Blechschmidt, I., Schroder, S., and Ambrosius, H. 1991. The MTT‐assay as a rapid test for cell proliferation and cell killing: Application to human peripheral blood lymphocytes (PBL). Allerg. Immunol. 37:139‐144.
   Wittstock, M., Rehfeldt, C., Mix, E., and Zettl, U.K. 2001. Comparison of three different proliferation assays for mouse myoblast cultures. Anal. Biochem. 292:166‐169.
   Zhi‐Jun, Y., Sriranganathan, N., Vaught, T., Arastu, S.K., and Ahmed, S.A. 1997. A dye‐based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies. J. Immunol. Methods 210:25‐39.
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