Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM)

Abstract

In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following pulse-labelling with 5'-bromo-2'-deoxyuridine (BrdUrd), cytocentrifuged, fixed in ethanol, and then exposed to UV light to induce DNA strand breaks at the sites of incorporated BrdUrd. The 3' OH termini of the photolytically generated DNA strand breaks were labelled with BrdUTP in the reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), followed by FITC-labelled BrdUrd antibodies. DNA was counterstained with propidium iodide (PI). Due to differences in chromatin structure between the interphase and mitotic cells, the LSC identified the latter by virtue of their higher red (PI) fluorescence intensity values among all pixels over the measured cell. To confirm that the cells selected were indeed cells in mitosis, predominantly in metaphase, the recorded X-Y coordinates of selected cells were used to re-position the cell for their visual examination. From the time lapse analysis of percentage BrdUrd-labelled cells progressing through mitosis it was possible to calculate the duration of individual phases of the cell cycle. The duration of S (Ts) and G2 + M (TG2 + M) was 8 and 3 h, respectively, and the minimal duration of G2 (TG2) was 2 h. The cell cycle time (Tc) estimated for the cohort of the most rapidly progressing cells was 13 h. The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis.

Demethoxycurcumin induces the apoptosis of human lung cancer NCI-H460 cells through the mitochondrial-dependent pathway

“…Cell viability was estimated through flow cytometric methods as described elsewhere (14,15). (16,17). The cell cycle distribution and sub-G1 groups (apoptosis) were calculated and analyzed by CellQuest (Becton-Dickinson) and ModFit LT software (Verity Software House Inc., Topsham, ME, USA).…”

“…5 cells/well) were placed on 6-well chamber slides and incubated with or without 35 µM DMC for 48 h. The cells were then fixed, washed and permeabilized as described previously (16,17). Then anti-AIF, Endo G, ATF6β, IRE1α and p-PERK (all in green fluorescence) were individually used for staining each sample overnight, followed by washing and then staining with the secondary antibody (FITC-conjugated goat anti-mouse IgG).…”

“…The cells were then incubated for 0, 6, 24 and 48 h and harvested, washed and resuspended in 25 µl of 10 µM substrate solution (PhiPhiLux and CaspaLux kit; OncoImmunin, Inc., Gaithersburg, MD, USA) before being incubated at 37˚C for 60 min. The cells were washed again in PBS and were analyzed by flow cytometry as described previously (16,18,19).…”

“…At the end of the incubation, cells from each treatment and time-points were collected, washed, counted and then were resuspended in 500 µl of DCFH-DA (10 µM) for 30 min for ROS (H 2 O 2 ) measurement, resuspended in 500 µl of Fluo-3/AM (2.5 µg/ml) for 30 min for intracellular Ca 2+ concentration measurement and resuspended in 500 µl of DiOC 6 (4 µmol/l) for 30 min to determine the levels of ΔΨm. All samples were then individually analyzed by flow cytometry as described previously (16).…”

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes

“…Human promyelocytic leukemic HL-60 cells (obtained from the American Type Culture Collection, Rockville, MD) were cultured in the medium as above, and during their exponential growth phase were treated with 150 nM DNA topoisomerase I inhibitor camptothecin (CPT; Sigma Chemical Co., St. Louis, MO) for 2 or 4 h. All tissue culture media and reagents, including PHA, were obtained from Gibco/BRL Life Technologies, Inc. (Grand Island, NY). Other details of lymphocyte and HL-60 culturing are provided elsewhere (21,32).…”