Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death

• Published in: Apoptosis (London) Vol. 24; no. 5-6; pp. 453 - 464
• Main Authors: Bergamaschi, D., Vossenkamper, A., Lee, W. Y. J., Wang, P., Bochukova, E., Warnes, G.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-06-2019; Springer; Springer Nature B.V
• Subjects: Analysis; Apoptosis; Autophagy; Biochemistry; Biomarkers - metabolism; Biomedical and Life Sciences; Biomedicine; Cancer Research; Caspase; Caspase 3 - metabolism; Caspase Inhibitors - pharmacology; Caspase-3; Cell Biology; Cell death; Cell Line; Cell Survival - drug effects; Chloroquine; Combined treatment; Deoxyribonucleic acid; DNA; DNA Damage; Flow Cytometry; Humans; Imidazoles - pharmacology; Immunophenotyping; Indoles - pharmacology; Intracellular; Jurkat Cells; K562 Cells; Mortality; Naphthoquinones - pharmacology; Necroptosis; Oncology; Phagocytosis; Phenotypes; Populations; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; Regulated Cell Death - drug effects; Shikonin; Virology; RIP1-dependent apoptosis; Flow cytometry; Hyper-activation of PARP; Necroptosis; DNA damage response; Regulated cell death; Autophagy; Parthanatos; Apoptosis
• ISSN: 1360-8185; 1573-675X
• DOI: 10.1007/s10495-019-01528-w

Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3
+ ve
/RIP3
− ve
), necroptosis (RIP3
Hi + ve
/Caspase-3
− ve
) and RIP1-dependent apoptosis (Caspase-3
+ ve
/RIP3
+ ve
) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations. Shikonin simultaneously induced Jurkat cell apoptosis and necroptosis the degree of which can be shown flow cytometrically together with the effects of blockade of these forms of cell death by zVAD and necrostatin-1 have on specific RCD populations including necroptosis, early and late apoptosis and RIP1-dependent apoptosis phenotypes in live and dead cells. Necrostatin-1 and zVAD was shown to modulate levels of shikonin induced DDR, hyper-action of PARP and parthanatos in the four forms of RCD processes analysed. LC3B was up-regulated by combined treatment of zVAD with chloroquine which also revealed that DNA damage was reduced in live cells but enhanced in dead cells indicating the role of autophagy in maintaining cell health. This approach to RCD research should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations.