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. 2024 Jun 25;10(13):e33665.
doi: 10.1016/j.heliyon.2024.e33665. eCollection 2024 Jul 15.

Flow cytometry-based quantitative analysis of cellular protein expression in apoptosis subpopulations: A protocol

Salah Abdalrazak Alshehade  1 , Hassan A Almoustafa  2   3 , Mohammed Abdullah Alshawsh  2   4 , Zamri Chik  2   3
Affiliations

Flow cytometry-based quantitative analysis of cellular protein expression in apoptosis subpopulations: A protocol

Salah Abdalrazak Alshehade et al. Heliyon. .

Abstract

Flow cytometry techniques utilizing dual staining with annexin V and propidium iodide (PI) provide a robust method for quantitatively analyzing apoptosis induction. Annexin V binds phosphatidylserine exposed on the outer leaflet of the plasma membrane during early apoptosis, while PI permeates late apoptotic/necrotic cells. Simultaneous staining allows differentiation of viable, early apoptotic, and late apoptotic/necrotic populations. This approach can be enhanced by using fluorochrome-conjugated antibodies to stain specific proteins, enabling the simultaneous tracking of protein expression changes in defined cell subpopulations during apoptosis. This multiparametric approach provides key insights into signaling regulation and the mechanisms underlying the apoptotic response to cytotoxic treatments. Here we present a protocol that combines annexin V-FITC/PI staining with APC-conjugated antibody labeling in MDA-MB-231 breast cancer cells treated with doxorubicin. This protocol enables both the quantitative assessment of apoptosis induction and the tracking of decreased CD44 expression from viable to apoptotic cells. This protocol also provides guidelines for appropriate filter selection, compensation controls, gating strategies, and troubleshooting. This robust protocol holds significant potential for elucidating signaling networks involved in apoptosis and therapeutic resistance across various cellular models.

Keywords: Annexin V; Apoptosis; Flow cytometry; Propidium iodide; Protein expression.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Annexin V-FITC/PI analysis of MDA-MB-231 breast cancer cells treated with 1 μM doxorubicin for 72 h and stained for surface CD44 and CD24 expression using APC-anti-CD44 and APC-anti-CD24 antibodies. Quadrants showing Annexin V/PI fluorescence are numbered as follows: Healthy cells (Annexin V-/PI-), Early apoptotic (Annexin V+/PI-), Late apoptotic (Annexin V+/PI+), and necrotic (Annexin V-/PI+) cells.
Fig. 2
Fig. 2
MDA-MB-231 breast cancer cells treated with 1 μM doxorubicin for 72 h and stained for surface CD24 (A) and CD44 (B) expression among different apoptosis subpopulations. *p < 0.05, ***p < 0.001.
Fig. 3
Fig. 3
Histogram of MDA-MB-231 breast cancer cells treated with 1 μM doxorubicin for 72 h and stained for surface CD44 (A) and CD24 (C) expression among different apoptosis subpopulations (B and D).
Fig. 4
Fig. 4
Histogram of MDA-MB-231 breast cancer untreated cells and stained for surface CD44 (A) and CD24 (C) expression among different apoptosis subpopulations (B and D).
See this image and copyright information in PMC

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