Cell cycle distribution was measured using a Cycle TEST™ PLUS DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA) and following the manufacturer’s protocol. PANC-1 cells were seeded at a concentration of 1 × 105 cells/mL and left overnight in an incubator. Prior to the treatment, the cells were serum-starved for 24 h and were treated accordingly the next day. Briefly, the floating and adherent cells were collected, spun and washed with 1X cold PBS. Then, the concentrations of cells were adjusted to 1 × 106 cells/mL and stained with propidium iodide (BD Biosciences) in the presence of RNAse buffer (BD Biosciences) for 30 min on ice and in a dark condition. The results of staining with 15,000 cells per sample were then collected by flow cytometry (Becton Dickinson FacsCalibur, Franklin Lakes, NJ, USA) and analyzed using Modfit Lt 5.0 software (Becton Dickinson).
Modfit lt 5
Manufactured by BD
Sourced in United States
Modfit Lt 5.0 is a software application designed for data analysis and processing. It provides a suite of tools for the management and interpretation of flow cytometry data.
Automatically generated - may contain errors
3 protocols using modfit lt 5
1
Cell Cycle Analysis of PANC-1 Cells
2
Inducible Cre-mediated Recombination
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Ad293 cells seeded overnight in 96-wells were transfected with pCMV-CreER, pCMV-mTEVp, pCMV-mCreER, or pCMV-mCreER/pCMV-mTEVp. Rapamycin and/or tamoxifen were added at 24 h after transfection. Cells were collected after 24 h or 48 h induction. Samples from three 96-wells were pooled and applied to a BD FACSAria (Becton Dickinson, Franklin Lakes, NJ) equipped with a 70-mm nozzle. Dead cells and debris were excluded using FSC (forward scatter) and SSC (side scatter). A total of 10,000 events for each sample were acquired and analyzed with Flow Jo VX 10. Recombination rate was calculated as the percentage of EGFP-positive cells vs. total fluorescently positive cells. Leakiness was calculated as the ratio between the uninduced and the induced recombination rate.
DNA content was determined using the Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China). Cells from three 96-wells were harvested, washed one time with ice-cold PBS, and dispersed gently by vortexing. We added 1 mL ice-cold 70% ethanol drop by drop. After fixing at 4°C overnight, cells were incubated in staining solution containing 50μg/mL propidium iodine and 20 μg/mL RNase A at 37°C for 30 min. The sample was run in a BD FACSAria (Becton Dickinson). After exclusion of doublets, the analysis of cell cycle distribution was performed with ModFit LT 5.0 Software (Becton Dickinson).
DNA content was determined using the Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China). Cells from three 96-wells were harvested, washed one time with ice-cold PBS, and dispersed gently by vortexing. We added 1 mL ice-cold 70% ethanol drop by drop. After fixing at 4°C overnight, cells were incubated in staining solution containing 50μg/mL propidium iodine and 20 μg/mL RNase A at 37°C for 30 min. The sample was run in a BD FACSAria (Becton Dickinson). After exclusion of doublets, the analysis of cell cycle distribution was performed with ModFit LT 5.0 Software (Becton Dickinson).
3
Cell Cycle Analysis of Kidney Tumor Cells
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After infected with target virus, cell cycle distribution was investigated to evaluate the effect of KPNA2 on kidney tumour cell. Briefly, the cells were made into cell suspensions using trypsin and collected in a 5‐mL centrifuge tube. The cells were centrifuged at 1300 rpm for 5 minutes, and then, the supernatant was discarded. Then, the cells were washed by D‐Hanks (pH 7.2–7.4). The cells were centrifuged at 1300 rpm for 5 minutes once again and then fixed using 75% ethanol for at least 1 hour. The cells were centrifuged at 1300 rpm for 5 minutes and then washed by D‐Hanks (pH 7.2–7.4) once again. 40 × propidium iodide and 100 × RNase were fixed and added to the cells (0.6–1 mL). Cell cycles of these cells were then examined by flow cytometer (Millipore, Darmstadt, Germany). In addition, the analysis was performed with ModFit LT 5.0 software (Becton Dickinson).
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