Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation

Abstract

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G/G, sub-S and sub-G/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.

G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress

“…Arsenite has also been shown to delay progression through the cell cycle and to induce apoptosis following G2/M Arrest ( 54 , 55 ). Accordingly, arsenite induced cell cycle arrest, as determined by FACS analysis (Figure 2E ), in agreement with our pathway analysis and as reported ( 56 ).…”

(2E)-2-Benzylidene-4,7-dimethyl-2,3-dihydro-1H-inden-1-one (MLT-401), a novel arylidene indanone derivative, scavenges free radicals and exhibits antiproliferative activity of Jurkat cells

“…Although apoptosis is a distinct form of programmed cell death, which is usually defective in cancerous cells, the ladder formation we observed is indicative of induced apoptosis [31]. The sub-G 0 /G 1 cell cycle fraction accumulation further supports induced apoptosis in the Jurkat cells [32]. The oxidant scavenging activity of MLT-401 may be responsible for the gross apoptosis in the Jurkat cells because it alters the malignant physiology of the cells compelling them to undergo apoptosis.…”

Prediction of Spheroid Cell Death Using Fluorescence Staining and Convolutional Neural Networks

“…The model obtained from this study can predict live and dead spheroids using fluorescence staining dyes, including PI and CA. PI can distinguish between live cells and late-stage apoptosis/necrosis cells, 28 allowing the model to predict a spheroid as dead if it is within the late-stage apoptosis/necrosis cell cycle. Moreover, CA can distinguish the metabolic activity between live and dead cells using the esterase enzyme, 29 enabling this model to predict a spheroid as live based on intracellular esterase activity.…”