Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Abstract

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.

Biological evaluation of 5-fluorouracil nanoparticles for cancer chemotherapy and its dependence on the carrier, PLGA

“…Thus, live cells show a normal green nucleus; early apoptotic cells a bright green nucleus with condensed or fragmented chromatin; late apoptotic cells show condensed and fragmented orange chromatin; and cells that have died from direct necrosis show a structurally normal orange nucleus. 27,28 Our results of AO/ EB staining also exhibited a higher number of apoptotic cells on treatment with PLGA 50-50 nanoformulation ( Figures 10 and 11) compared with free 5-FU and PLGA 90-10 nanoparticles. Since 5-FU interferes with DNA synthesis, 41 the alterations in cell cycle progression on treatment with 5-FU formulations were studied using fluorescence-activated cell sorting (FACS) analysis.…”

“…Cell viability (%) related to control wells containing cell culture medium without treatment was calculated by [A] AO/eB assay AO/EB assay is reported to be a reliable method for the quantification of apoptosis. 27,28 Thus, for fluorescence cytochemical study, cells were seeded in a 35 mm culture dish and 5-FU treatment (100 µM) was given for 24 hours. Medium was removed, and the cells were washed with PBS.…”

Overexpression of CYP2E1 enhances sensitivity of HepG2 cells to Fas-mediated cytotoxicity

“…AO/PI double staining was carried out as previously described 22 with some modifications. Briefly, cells were split and palletized by centrifuging.…”

Sulfur dioxide attenuates LPS-induced acute lung injury via enhancing polymorphonuclear neutrophil apoptosis

“…A fluorescent assay with AO/EB double staining was used [20] to display the morphological alterations of apoptotic PMNs in BALF. The BALF PMNs were suspended in PBS (pH 7.2) and aliquoted for the staining procedure.…”

“…Peripheral blood PMN cell culture and evaluation of PMN apoptosis by FCM Peripheral blood PMNs were cultured in RPMI-1640 medium at 37 °c in 5% cO 2 and treated with 30 mg/L LPS and different concentration of SO 2 (10,20, and 30 µmol/L SO 2 ) in vitro for 6 h for the detection of apoptosis-related protein expression using the Western blotting method. To determine the rate of apoptosis, PMNs were cultured for 24 h and then submitted to FCM.…”