Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane

Abstract

Background:Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria‐dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time‐consuming and inaccurate, and the latter is still limited by sample size.Methods:We developed a rapid and reliable technique to detect cytochrome c release during drug‐induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL‐60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells.Results:The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4–8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c.Conclusions:This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose. © 2006 International Society for Analytical Cytology

Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis

“…After two washes with PBS, cells were incubated in 0.5 ml of labeling medium (2% fetal bovine serum, 0.2% sodium azide, and 0.5% Triton X-100 in PBS) for 15 min, centrifuged at 3000 g for 5 min, and then incubated with anti-cytochrome c (1:500, 6 H2.B4, Promega, USA) at 41C for 1 h. Cells were then washed twice in the same medium and incubated with anti-mouse-FITC (1:200, Vector Laboratories, USA) at 41C for 1 h. Cells were washed again as described above, resuspended in PBS, and analyzed by flow cytometry as described elsewhere. 38 Detection of Caspase-3 Activation Caspase-3 activation was measured by the incubation of 10 6 cells with FITC-DEVD-FMK (1:300, Calbiochem, USA) in serum-free medium for 40 min at 371C in a humidified atmosphere with 5% CO 2 . After a washing step according to the manufacturer instructions, cells were resuspended in the same medium and analyzed by flow cytometry, as described before.…”

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

“…Cytochrome c release Release of cytochrome c, a putative event of the mitochondria apoptotic pathway following loss of Δψm, was measured as described [52,53]. After H 2 O 2 treatment, the cells were washed in PBS, resuspended in 1 ml of mitochondrial medium containing 250 mM sucrose, 10 mM KCl, 20 mM HEPES-KOH, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1.5 mM MgCl 2 , and 1% protease inhibitors mixture.…”

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

“…Such assays are commercially available for the detection of Cyt c and ensure a high degree of sensitivity [67,68]. Recently, a cytofluorometric method for the immunodetection of Cyt c following the digitonin-mediated permeabilization of plasma membrane has been proposed [69]. In the first step of the assay, plasma membranes (but not mitochondrial membranes) are permeabilized by low concentrations of digitonin, thus causing the wash out of cytosolic (but not [69].…”

“…Recently, a cytofluorometric method for the immunodetection of Cyt c following the digitonin-mediated permeabilization of plasma membrane has been proposed [69]. In the first step of the assay, plasma membranes (but not mitochondrial membranes) are permeabilized by low concentrations of digitonin, thus causing the wash out of cytosolic (but not [69]. Despite the theoretical possibility to process a large number of samples, this technique relies on a sophisticated permeabilization protocol, which may require repeated optimization (for instance depending on the cell type and on the digitonin stock).…”