Flow Cytometry of Apoptosis

Abstract

Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. Early events of apoptosis, dissipation of the mitochondrial transmembrane potential and caspase activation, can be detected using either fluorochrome reporter groups or appropriate antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V. Another apoptotic event, DNA fragmentation based on DNA content of cells with fractional (“sub‐G1”) or DNA strand‐break labeling, TUNEL; or In Situ End Labeling, ISEL;. Still another hallmark of apoptosis is the activation of tissue transglutaminase (TGase), the enzyme that crosslinks protein and thereby makes them less immunogenic. The major advantage of flow cytometry in these applications is that it provides the possibility of multiparametric measurements of cell attributes.

Identification of aurora kinase A as an unfavorable prognostic factor and potential treatment target for metastatic gastrointestinal stromal tumors

“…After treatment with MLN8237, the cells were washed once with 1x PBS and resuspended in 100 μl of staining solution (containing Annexin V-FITC and PI in a HEPES buffer). After being incubated at room temperature for 15 minutes, the cells were diluted in 1x Annexin V–binding buffer and the percentage of apoptotic cells was determined by flow cytometry FACS Calibur (Becton Dickinson and Co., Oxford, CA, USA) [48]. …”

Flow cytometric analysis of BDE 47 mediated injury to rainbow trout gill epithelial cells

“…As a result of DNA fragmentation and chromatin condensation, apoptotic cells show reduced DNA staining with PI, reflecting lower quantitative DNA content relative to G1 phase nuclei (Gong et al ., 1994). The shedding of apoptotic bodies containing fragments of nuclear chromatin may further contribute to the loss of DNA from apoptotic cells (Pozarowski et al ., 2004). Accordingly, the emergence of sub-G1 peaks seen with increasing levels of BDE 47 treatment indicates that these cells were undergoing apoptosis.…”

“…In this assay, apoptotic cells were identified on the basis of hypodiploid DNA content that results from DNA fragmentation (Nicoletti et al ., 1991; Pozarowski et al ., 2004). Cells having DNA content less than those cells in the G 1 phase of the cell cycle (sub-G1) were assumed to be apoptotic.…”

Vascular Endothelial Cell Growth Factor A Acts via Platelet-Derived Growth Factor Receptor α To Promote Viability of Cells Enduring Hypoxia

“…The percentage of apoptotic cells was determined by staining with FITC-conjugated annexin V. Cells were counterstained with propidium iodide (PI) to exclude nonviable cells. Annexin V-positive cells were measured from a total viable population of 50,000 cells by flow cytometry (17). The percentage of senescent cells was determined by quantifying senescence-associated ␤-galactosidase (SA-␤-Gal) activity using C 12 FDG, a ␤-Gal substrate that fluoresces upon being cleaved (18).…”

“…Retinal cell suspensions (prepared from explants or from freshly harvested retinas) were stained with PI to gate out nonviable cells, anti-PDGFR␣-phycoerythrin (PE) and anti-VEGFR2-CY5.5 to mark receptor-specific subpopulations, and DAPI for cell cycle analysis (19). Samples were then split 50:50: one 50% fraction received FITC-annexin V for apoptotic detection (17), and the other 50% fraction was stained with C 12 FDG, an FITC-based substrate for detecting senescent cells (18). Both halves were then analyzed in parallel for each treatment and condition.…”