HT29 Cells were harvested at 0, 24, 48 and 72 h after treatment and fixed with 70% ethanol at −20°C overnight. Cell pellets were washed once with PBS and DNA was stained using the Cycletest Plus DNA Reagent Kit (cat. no. 340242; BD Biosciences) following the manufacturer's instructions. The counts of cell cycle distribution were evaluated by PI staining, and all samples were analyzed using a FACSCanto II flow cytometer (BD Biosciences) at a wavelength of 488 nm with the appropriate software (BD FACSDiva Software ver. 8.0.2; BD Biosciences).
Facsdiva software ver 8
Manufactured by BD
Sourced in United States
The BD FACSDiva Software ver. 8.0.2 is a flow cytometry analysis software. It provides data acquisition, analysis, and visualization capabilities for flow cytometry experiments.
Automatically generated - may contain errors
4 protocols using facsdiva software ver 8
1
Cell Cycle Analysis of HT29 Cells
2
Cell Cycle Analysis of 5-FU and AZD6738 Treated HT29 Cells
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HT29 cells were treated with 5-FU and/or AZD6738 for 24 h at 37°C, followed by treatment with nocodazole (300 nM) for another 24 h at 37°C. Nocodazole was added to prevent cells from exiting mitosis. Subsequently, the cells were fixed with 70% ethanol at −20°C overnight. Cell pellets were washed once with PBS and stained with an antibody against phospho-histone H3 at S10 (dilution, 1:100; cat. no. 06–570; MilliporeSigma) for 3 h, followed by 30 min of incubation with an Alexa Fluor 488 secondary antibody (dilution, 1:50; cat. no. ab150077; Abcam). DNA was counterstained with a BD Cycletest Plus DNA Reagent Kit (cat. no. 340242; BD Biosciences). All samples were analyzed using a FACSCanto II flow cytometer (BD Biosciences) at a wavelength of 488 nm with appropriate software (BD FACSDiva Software ver. 8.0.2; BD Biosciences).
3
Flow Cytometry of Cell Suspensions
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Mewo-Luc cell suspensions just after PKH-labeling, and hMSC suspensions and sheets spiked with Mewo-Luc were subjected to FCM. FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA) and FACS Diva software ver. 8.0 (BD Biosciences) were used for analyses. Green phycoerythrin (PE) detector was used for the detection of PKH fluorescence. Mewo-Luc cell count included in a hMSC suspension or sheet based on FCM was calculated by total live cell number and live PKH-positive cell ratio measured by FCM.
4
Cell Cycle Analysis of Decidualizing HESCs
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Forty-eight hours following siRNA transfection with control siRNA or MBTD1 siRNA alone or PGR siRNA alone or both MBTD1 and PGR siRNAs together using Lipofectamine 2000 transfection agent (Invitrogen Corporation, Carlsbad, CA, United States), HESCs were trypsinized and counted. For flow cytometry analysis, 2 × 105 cells were plated per well of six-well plates and then were treated with decidualization (EPC) media. After cells were cultured in EPC media for 48 h, cells were trypsinized, washed with phosphate-buffered saline (PBS), fixed in 70% chilled ethanol, and stained with 50 μg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO, United States) containing 50 μg/ml RNase. Cell cycle stage analysis was performed by flow cytometry (FACS Canto II) and FACS Diva software (ver. 8.0; BD Biosciences, Franklin Lakes, NJ, United States). Each experiment was performed in technical duplicates and repeated in three independent HESC lines isolated from three subjects.
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