Apoptosis sometimes called "programmed cell death" is an orderly process by which cells die, as opposed to necrosis. Apoptosis can be determined using flow cytometry by a variety of methods. An early event of apoptosis is the externalization of phosphatidylserine on the cell membrane, which can be detected with the Annexin V protein. Apoptosis during T-cell development involves Fas and can be measured with the CD95 antibody. A later event in apoptosis is DNA fragmentation. DNA strand breaks can be labeled by the "tunel" method. DNA fragmentation can also lead to hypodiploid cells, which have a DNA content less than that for diploid G0/G1 cells.
This example comes from Dr. Milena Cankovic's work under Dr. Stephen Lerman to elucidate apoptosis in a murine model for human B-cell lymphoma. Murine splenocytes were exposed to irradiated tumor cells in a mixed lymphocyte interaction (MLI) assay. A gating strategy incorporating pulse processing parameters was used to eliminate aggregates (labeled "Doublets" above) and debris, leaving "Singlet Cells", from which CD4+ cells were selected. (CD4 is found primarily on T helper cells, which play an essential role in this lymphoma.) The CD4+ cells were divided into three subsets based on DNA content: G0/G1 (colored red above), hypodiploid (labeled "Apoptotic" and colored green above), and the few remainders which include S-, G2-, and M-phase cells (colored magenta above). This analysis was performed using Attractors* software to enumerate the fraction of CD4+ apoptotic cells. Apoptosis was confirmed by the "laddering" pattern seen by gel elecrophoresis.
*Attractors is a trademark of BD Biosciences.
CAUTION! Because in vivo samples like this one tend to have very few cells outside the G0- and G1-phases and low molecular weight DNA was extracted from cells undergoing apoptosis, the apoptotic cells show up as a distinct hypodiploid peak with DNA index between 0.5 and 1. For most in vitro samples, which typically contain a large number of cycling cells, this method is probably inappropriate and another method should be used instead. For example, mitotic cells undergoing apoptosis would be indistiguishable from normal G0-, G1-, and S-phase cells based on DNA content alone, since all of these cells would have DNA index between 1 and 2. (Furthermore, a particle originating from a mitotic cell that has undergone apoptosis and now has DNA index less than 1 would be an apoptotic body, not an apoptotic cell. Since more than one apoptotic body arises from one cell, it is important to only count apoptotic cells.) Also, as a general rule, apoptosis measured by flow cytometry should be confirmed by an independent assay, as above.
Yoshimura FK, Wang T, Nanua S. Mink cell focus-forming murine leukemia virus killing of mink cells involves apoptosis and superinfection. J Virol. 75(13):6007-15, 2001.
Kim HE, Krug MA, Han I, Ensley J, Yoo GH, Forman JD, Kim HR. Neutron radiation enhances cisplatin cytotoxicity independently of apoptosis in human head and neck carcinoma cells. Clin Cancer Res. 6(10):4142-7, 2000.
Nabha SM, Wall NR, Mohammad RM, Pettit GR, Al-Katib AM. Effects of combretastatin A-4 prodrug against a panel of malignant human B-lymphoid cell lines. Anticancer Drugs 11(5):385-92, 2000.
Tennenberg SD, Finkenauer R, Dwivedi A. Absence of lipopolysaccharide-induced inhibition of neutrophil apoptosis in patients with diabetes. Arch Surg. 134(11):1229-33,1999.
Glazyrin AL, Adsay VN, Vaitkevicius VK, Sarkar FH. CD95-related apoptotic machinery is functional in pancreatic cancer cells. Pancreas. 22(4):357-65, 2001.
Ali MA, Rosati R, Pettit GR, Kalemkerian GP. Dolastatin 15 induces apoptosis and BCL-2 phosphorylation in small cell lung cancer cell lines. Anticancer Res. 18(2A):1021-6, 1998.
Kalemkerian GP, Slusher R, Ramalingam S, Gadgeel S, Mabry M. Growth inhibition and induction of apoptosis by fenretinide in small-cell lung cancer cell lines. J Natl Cancer Inst. 87(22):1674-80, 1995.
Upadhyay S, Neburi M, Chinni SR, Alhasan S, Miller F, Sarkar FH. Differential sensitivity of normal and malignant breast epithelial cells to genistein is partly mediated by p21(WAF1). Clin Cancer Res. 7(6):1782-9, 2001.
Rosati R, Adil MR, Ali MA, Eliason J, Orosz A, Sebestyen F, Kalemkerian GP. Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer. Peptides. 19(9):1519-23, 1998.
Cankovic M, Van Buren E, Lerman S. CD4+ cell apoptosis in chemo/immunotherapy
of a T-helper-cell dependent B-cell lymphoma. The FASEB J. 8:
A745, 1994.
Van Buren E, Cankovic M, Lerman S. Advantages of apoptosis analysis
using the Attractors program. Cytometry 7: 53, 1994.