Skip to main content
Clinical and Diagnostic Laboratory Immunology logoLink to Clinical and Diagnostic Laboratory Immunology
. 1995 May;2(3):369–376. doi: 10.1128/cdli.2.3.369-376.1995

Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry.

T J Liegler 1, W Hyun 1, T S Yen 1, D P Stites 1
PMCID: PMC170162  PMID: 7664185

Abstract

Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.

Full Text

The Full Text of this article is available as a PDF (553.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akbar A. N., Borthwick N., Salmon M., Gombert W., Bofill M., Shamsadeen N., Pilling D., Pett S., Grundy J. E., Janossy G. The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory. J Exp Med. 1993 Aug 1;178(2):427–438. doi: 10.1084/jem.178.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arends M. J., Morris R. G., Wyllie A. H. Apoptosis. The role of the endonuclease. Am J Pathol. 1990 Mar;136(3):593–608. [PMC free article] [PubMed] [Google Scholar]
  3. Banda N. K., Bernier J., Kurahara D. K., Kurrle R., Haigwood N., Sekaly R. P., Finkel T. H. Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med. 1992 Oct 1;176(4):1099–1106. doi: 10.1084/jem.176.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carson D. A., Ribeiro J. M. Apoptosis and disease. Lancet. 1993 May 15;341(8855):1251–1254. doi: 10.1016/0140-6736(93)91154-e. [DOI] [PubMed] [Google Scholar]
  5. Chrest F. J., Buchholz M. A., Kim Y. H., Kwon T. K., Nordin A. A. Identification and quantitation of apoptotic cells following anti-CD3 activation of murine G0 T cells. Cytometry. 1993 Nov;14(8):883–890. doi: 10.1002/cyto.990140806. [DOI] [PubMed] [Google Scholar]
  6. Cohen G. M., Sun X. M., Snowden R. T., Dinsdale D., Skilleter D. N. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J. 1992 Sep 1;286(Pt 2):331–334. doi: 10.1042/bj2860331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen J. J., Duke R. C., Fadok V. A., Sellins K. S. Apoptosis and programmed cell death in immunity. Annu Rev Immunol. 1992;10:267–293. doi: 10.1146/annurev.iy.10.040192.001411. [DOI] [PubMed] [Google Scholar]
  8. Cohen J. J., Duke R. C. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol. 1984 Jan;132(1):38–42. [PubMed] [Google Scholar]
  9. Cohen J. J. Programmed cell death in the immune system. Adv Immunol. 1991;50:55–85. doi: 10.1016/s0065-2776(08)60822-6. [DOI] [PubMed] [Google Scholar]
  10. Collins R. J., Harmon B. V., Gobé G. C., Kerr J. F. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int J Radiat Biol. 1992 Apr;61(4):451–453. doi: 10.1080/09553009214551201. [DOI] [PubMed] [Google Scholar]
  11. Compton M. M., Haskill J. S., Cidlowski J. A. Analysis of glucocorticoid actions on rat thymocyte deoxyribonucleic acid by fluorescence-activated flow cytometry. Endocrinology. 1988 May;122(5):2158–2164. doi: 10.1210/endo-122-5-2158. [DOI] [PubMed] [Google Scholar]
  12. Dancescu M., Rubio-Trujillo M., Biron G., Bron D., Delespesse G., Sarfati M. Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression. J Exp Med. 1992 Nov 1;176(5):1319–1326. doi: 10.1084/jem.176.5.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Darzynkiewicz Z., Bruno S., Del Bino G., Gorczyca W., Hotz M. A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 1992;13(8):795–808. doi: 10.1002/cyto.990130802. [DOI] [PubMed] [Google Scholar]
  14. Del Bino G., Lassota P., Darzynkiewicz Z. The S-phase cytotoxicity of camptothecin. Exp Cell Res. 1991 Mar;193(1):27–35. doi: 10.1016/0014-4827(91)90534-2. [DOI] [PubMed] [Google Scholar]
  15. Dive C., Gregory C. D., Phipps D. J., Evans D. L., Milner A. E., Wyllie A. H. Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta. 1992 Feb 3;1133(3):275–285. doi: 10.1016/0167-4889(92)90048-g. [DOI] [PubMed] [Google Scholar]
  16. Duke R. C., Chervenak R., Cohen J. J. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6361–6365. doi: 10.1073/pnas.80.20.6361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  18. Gorczyca W., Gong J., Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 1993 Apr 15;53(8):1945–1951. [PubMed] [Google Scholar]
  19. Gougeon M. L., Olivier R., Garcia S., Guetard D., Dragic T., Dauguet C., Montagnier L. Mise en évidence d'un processus d'engagement vers la mort cellulaire par apoptose dans les lymphocytes de patients infectés par le VIH. C R Acad Sci III. 1991;312(11):529–537. [PubMed] [Google Scholar]
  20. Groux H., Torpier G., Monté D., Mouton Y., Capron A., Ameisen J. C. Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J Exp Med. 1992 Feb 1;175(2):331–340. doi: 10.1084/jem.175.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hardin J. A., Sherr D. H., DeMaria M., Lopez P. A. A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation. J Immunol Methods. 1992 Sep 18;154(1):99–107. doi: 10.1016/0022-1759(92)90217-h. [DOI] [PubMed] [Google Scholar]
  22. Henderson S., Rowe M., Gregory C., Croom-Carter D., Wang F., Longnecker R., Kieff E., Rickinson A. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell. 1991 Jun 28;65(7):1107–1115. doi: 10.1016/0092-8674(91)90007-l. [DOI] [PubMed] [Google Scholar]
  23. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  24. Hotz M. A., Gong J., Traganos F., Darzynkiewicz Z. Flow cytometric detection of apoptosis: comparison of the assays of in situ DNA degradation and chromatin changes. Cytometry. 1994 Mar 1;15(3):237–244. doi: 10.1002/cyto.990150309. [DOI] [PubMed] [Google Scholar]
  25. Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug;26(4):239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kubbies M. Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding. Cytometry. 1990;11(3):386–394. doi: 10.1002/cyto.990110309. [DOI] [PubMed] [Google Scholar]
  27. Lyons A. B., Samuel K., Sanderson A., Maddy A. H. Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry. Cytometry. 1992;13(8):809–821. doi: 10.1002/cyto.990130803. [DOI] [PubMed] [Google Scholar]
  28. Meyaard L., Otto S. A., Jonker R. R., Mijnster M. J., Keet R. P., Miedema F. Programmed death of T cells in HIV-1 infection. Science. 1992 Jul 10;257(5067):217–219. doi: 10.1126/science.1352911. [DOI] [PubMed] [Google Scholar]
  29. Newell M. K., Haughn L. J., Maroun C. R., Julius M. H. Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen. Nature. 1990 Sep 20;347(6290):286–289. doi: 10.1038/347286a0. [DOI] [PubMed] [Google Scholar]
  30. Nicoletti I., Migliorati G., Pagliacci M. C., Grignani F., Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991 Jun 3;139(2):271–279. doi: 10.1016/0022-1759(91)90198-o. [DOI] [PubMed] [Google Scholar]
  31. Ormerod M. G., Collins M. K., Rodriguez-Tarduchy G., Robertson D. Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. J Immunol Methods. 1992 Aug 30;153(1-2):57–65. doi: 10.1016/0022-1759(92)90305-d. [DOI] [PubMed] [Google Scholar]
  32. Ormerod M. G., Sun X. M., Snowden R. T., Davies R., Fearnhead H., Cohen G. M. Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. Cytometry. 1993;14(6):595–602. doi: 10.1002/cyto.990140603. [DOI] [PubMed] [Google Scholar]
  33. Payne C. M., Bjore C. G., Jr, Schultz D. A. Change in the frequency of apoptosis after low- and high-dose X-irradiation of human lymphocytes. J Leukoc Biol. 1992 Oct;52(4):433–440. doi: 10.1002/jlb.52.4.433. [DOI] [PubMed] [Google Scholar]
  34. Schmid I., Uittenbogaart C. H., Giorgi J. V. Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry. 1994 Jan 1;15(1):12–20. doi: 10.1002/cyto.990150104. [DOI] [PubMed] [Google Scholar]
  35. Schmid I., Uittenbogaart C. H., Keld B., Giorgi J. V. A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J Immunol Methods. 1994 Apr 15;170(2):145–157. doi: 10.1016/0022-1759(94)90390-5. [DOI] [PubMed] [Google Scholar]
  36. Sellins K. S., Cohen J. J. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol. 1987 Nov 15;139(10):3199–3206. [PubMed] [Google Scholar]
  37. Sun X. M., Snowden R. T., Skilleter D. N., Dinsdale D., Ormerod M. G., Cohen G. M. A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Anal Biochem. 1992 Aug 1;204(2):351–356. doi: 10.1016/0003-2697(92)90251-2. [DOI] [PubMed] [Google Scholar]
  38. Swat W., Ignatowicz L., Kisielow P. Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry. J Immunol Methods. 1991 Mar 1;137(1):79–87. doi: 10.1016/0022-1759(91)90396-w. [DOI] [PubMed] [Google Scholar]
  39. Telford W. G., King L. E., Fraker P. J. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry. 1992;13(2):137–143. doi: 10.1002/cyto.990130205. [DOI] [PubMed] [Google Scholar]
  40. Terai C., Kornbluth R. S., Pauza C. D., Richman D. D., Carson D. A. Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1. J Clin Invest. 1991 May;87(5):1710–1715. doi: 10.1172/JCI115188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  42. Visser J. W., van den Engh G. J., van Bekkum D. W. Light scattering properties of murine hemopoietic cells. Blood Cells. 1980;6(3):391–407. [PubMed] [Google Scholar]
  43. Watanabe-Fukunaga R., Brannan C. I., Copeland N. G., Jenkins N. A., Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature. 1992 Mar 26;356(6367):314–317. doi: 10.1038/356314a0. [DOI] [PubMed] [Google Scholar]
  44. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]
  45. Wyllie A. H., Rose K. A., Morris R. G., Steel C. M., Foster E., Spandidos D. A. Rodent fibroblast tumours expressing human myc and ras genes: growth, metastasis and endogenous oncogene expression. Br J Cancer. 1987 Sep;56(3):251–259. doi: 10.1038/bjc.1987.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zamai L., Falcieri E., Zauli G., Cataldi A., Vitale M. Optimal detection of apoptosis by flow cytometry depends on cell morphology. Cytometry. 1993 Nov;14(8):891–897. doi: 10.1002/cyto.990140807. [DOI] [PubMed] [Google Scholar]

Articles from Clinical and Diagnostic Laboratory Immunology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES