Cells (1×106 cells/well) were seeded on 6 well plates (Corning Life Sciences, Shanghai, China) and cultured with DMEM medium at 37°C with 5% CO2 for 24 h, prior to being transfected with 100 nM HSP70-siRNA or control siRNA (siCON) for 48 h. For the flow cytometry, cells were trypsinized, pelleted via centrifugation at 1,000 × g for 5 min at 4°C and resuspended in 300 µl 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)/PBS. Cells were fixed with the cold 70% ethanol (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 2 h and centrifuged at 1,000 × g for 5 min at 4°C. Subsequent to washing twice in cold PBS and centrifugation at 1,000 × g for 5 min at 4°C, cells were treated with RNase Type I-A (Sigma-Aldrich; Merck KGaA) at 37°C for 15 min and stained with PI (1 mg/ml) at room temperature for 10 min in the dark. Cellular DNA content was determined using a FACSCalibur™ (BD Biosciences). Cell cycle phase distributions were analyzed by ModFit LT™ (v3.0; BD Biosciences) cell cycle analysis software.
Modfit lt
Manufactured by BD
Sourced in United States
ModFit LT is a cell cycle analysis software used for analyzing flow cytometry data. It provides tools for DNA content analysis, cell cycle modeling, and data visualization. The software is designed to offer a user-friendly interface and advanced analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (6 mentions)
Cellquest, by BD (5 mentions)
Rnase a, by Merck Group (3 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
6 well plate, by Corning (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Flow cytometry, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Rnase type 1 a, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ethanol, by Sangon (1 mentions)
M6494, by Thermo Fisher Scientific (1 mentions)
Victor multilabel plate reader, by PerkinElmer (1 mentions)
Facsverse, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Culture insert, by BD (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Annexin 5 pe and 7 aad staining, by BD (1 mentions)
23 protocols using modfit lt
1
Cell Cycle Analysis of HSP70-siRNA Transfection
2
Cell Proliferation and Cell Cycle Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell proliferation analyses, cells were seeded in octuplicate into 96-well plates at a density of 2.5 × 103 cells per well and the experiment was repeated three times (independent biological replicates). After incubation for 3, 6, 9, and 12 days with either drugs or vehicles, cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M6494, Thermofisher Scientific) at a final concentration of 1 mg/mL, according to the manufacturer’s instructions. Absorbance was measured at 570 and 620 nm (background) wavelengths by the VICTOR Multilabel Plate Reader (PerkinElmer, Milan, Italy).
For cell cycle analysis, 105 cells were incubated with increasing concentrations of a compound for 3, 6, 9, or 12 days with ICI 182,780, EPZ004777 or the vehicle, by refreshing every 3 days. All assays were carried out in triplicate.
Cells were then fixed in cold 70% ethanol at 4 °C, treated with RNase A (10 μg/mL) for 10 min at room temperature, and stained with 20 μg/mL Propidium iodide (Sigma-Aldrich) for 30 min. Flow cytometry was performed using a BD FACSVerse™ (BD Biosciences, San Josè, CA) and data processed using the software ModFit LT™ (version 5.0, company, city, country). Results shown were obtained from three independent experiments.
For cell cycle analysis, 105 cells were incubated with increasing concentrations of a compound for 3, 6, 9, or 12 days with ICI 182,780, EPZ004777 or the vehicle, by refreshing every 3 days. All assays were carried out in triplicate.
Cells were then fixed in cold 70% ethanol at 4 °C, treated with RNase A (10 μg/mL) for 10 min at room temperature, and stained with 20 μg/mL Propidium iodide (Sigma-Aldrich) for 30 min. Flow cytometry was performed using a BD FACSVerse™ (BD Biosciences, San Josè, CA) and data processed using the software ModFit LT™ (version 5.0, company, city, country). Results shown were obtained from three independent experiments.
3
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometric analysis of the cell cycle, FACS analysis was performed as described below: after 24 h incubation, cells were washed with PBS and incubated with 0.25% trypsin for 5 min at 37 °C. After centrifuging for 5 min at 1000× g cells were washed with PBS, resuspended in 0.9 mL PBS, then 2.1 mL of 100% EtOH was added, and cells were vortexed. Cells were incubated for 40 min on ice. After washing cells twice with PBS, 1 mL RNse A solution was added, and cells were incubated for 30 min at 37 °C. Cells were then centrifuged for 5 min at 1000× g, 1 mL PI solution was added, and cells were incubated for 20 min at room temperature (RT).
The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACSCalibur (BD) with appropriate software (ModFit LT; BD, Topsham, ME, USA).
All experiments were performed using more than three individual trials and the significance of any intergroup differences was determined by ANOVA.
The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACSCalibur (BD) with appropriate software (ModFit LT; BD, Topsham, ME, USA).
All experiments were performed using more than three individual trials and the significance of any intergroup differences was determined by ANOVA.
4
Cell Cycle Progression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the cell cycle progression, the cells were seeded into culture dishes (1 × 106 cells), incubated for 24 h to allow exponential growth, and then they were treated with ISL at the indicated concentrations. All the cells were collected, slowly added to 9 mL 70% ethanol, and then they were stored at −20 °C for at least 2 h. Then, the cells were washed at least once with cold phosphate-buffered saline (PBS), resuspended in 300–500 µL PI/Triton X-100 staining solution (10 mL 0.1% (v/v) Triton X-100 in PBS containing 2 mg DNAse-free RNAse A and 0.40 mL of 500 µg/mL PI), and incubated for 30 min at 20 °C. The fluorescence was measured using a fluorescence-activated cell-sorting (FACS) Calibur flow cytometer (BD, San Jose, CA, USA) and the cell cycle distribution was analyzed using CellQuest and Modfit LT programs (BD).
5
Cell Proliferation, Apoptosis, and Transwell Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell proliferation activity was assessed by EdU (5-ethynyl-2′-deoxyuridine) incorporation and flow cytometry. The EdU assay was performed according to the manufacturer′s instructions (RiboBio, Guangzhou, China), and the percentage of EdU-positive cells was used to evaluate cell proliferation. For flow cytometry, the cells were stained with 2 µM CFSE (Sigma, USA). Data were analyzed using Cell Quest and ModFitLT software (BD, USA), and the proliferation index was calculated as previously described31 (link). For apoptosis, the cells were assayed using a FITC AnnexinV Apoptosis Detection Kit (BD, USA) followed by flow cytometry analysis. For in vitro transwell assays, 1 × 105 cells were seeded in the upper chamber of a culture insert (8-mm pore size in 24-well plates, BD, USA) without (migration assay) or with (invasion assay) Matrigel (BD, USA) in serum-free media. The bottom chamber was filled with the same medium supplemented with 10% fetal bovine serum. After 24 h, the cells on the undersurface of the membrane were stained with crystal violet and counted using a light microscope. The cell migration and invasion properties were evaluated by the number of cells per high power field. All experiments were performed in triplicate and with at least three biological replicates.
6
Cell Cycle Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The manufacturer’s instructions (MULTI SCIENCES, Hangzhou, China) were followed. A cell suspension was collected, and the number of cells ranged from 2×10
5 to 1×10
6 cells/mL. The cells were then centrifuged, and the supernatant was removed. PBS was used to wash the cells once, and the supernatant was discarded. Then, 1 mL of reagent A and 10 μL of reagent B were added to the cells, which were vortexed and mixed for 5‒10 s. The mixture was incubated at room temperature for 30 min. Finally, the percentages of cell cycle distribution were evaluated through PI staining, and all samples were analyzed using FACSCalibur (BD, Franklin Lakes, USA) with appropriate software (ModFit LT; BD).
5 to 1×10
6 cells/mL. The cells were then centrifuged, and the supernatant was removed. PBS was used to wash the cells once, and the supernatant was discarded. Then, 1 mL of reagent A and 10 μL of reagent B were added to the cells, which were vortexed and mixed for 5‒10 s. The mixture was incubated at room temperature for 30 min. Finally, the percentages of cell cycle distribution were evaluated through PI staining, and all samples were analyzed using FACSCalibur (BD, Franklin Lakes, USA) with appropriate software (ModFit LT; BD).
7
Cell Cycle Analysis of MDA-MB-231 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 cells were seeded and cultured overnight in 6-well plates. After treatment, cells were harvested, washed twice with ice cold PBS (pH 7.4) and fixed in 70 % ethanol for overnight at 4 °C. Then, cells were incubated with 250 μl of RNase A (100 μg/ml) for 30 min at 37 °C and finally stained with 500 μl of PI (50 μg/ml) for 1 h in the dark. Stained cells were analyzed with a BD LSRFortessa Cell Analyzer. Three independent experiments were performed. The relative percentages of cells in G1, S, or G2/M phase were calculated from FL-2 histograms using appropriate software (ModFit LT; BD, Topsham, ME, USA).
8
Cell Cycle and Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The percentages of cell cycle distribution were evaluated by PI staining. The cells were washed with precooled PBS and fixed overnight with 70% ethanol at 4°C. After rewashing with precooled PBS, 5% propidium iodide (Beyotime, China) was used for labelling the cells and incubated at 37℃ in dark for 30 min. The cell cycle was then analyzed by flow cytometer. Apotosis detected using an Annexin V fluorescein isothiocyanate kit according to the protocol. The cells were resuscitated with 100 μL 1× binding buffer, and the cell concentration was kept at about 1 × 106 cells /mL. Annexin V-PE and 7-AAD staining (BD) were added to the 1× binding buffer to prepare the cell staining solution (Annexin V-PE: 7-AAD: 1× binding buffer = 1:1:20). The cells were thereafter stained in the dark for 15 min, and analyzed by flow cytometry within 1 h. All samples were analyzed employing FACSCalibur (BD) with appropriate software (ModFit LT; BD, Topsham, ME) and the FlowJo (Ashland, OR) software were used for data analysis.
9
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell populations were treated with erufosine, washed with PBS and fixed in 70% ethanol-PBS solution for 1 h on ice. Fixed cells were washed twice with ice cold PBS, treated with RNAse A for 30 min at 37°C and stained with propidium iodide 15 min prior to analysis. Cellular DNA content was determined by flow cytometry using the DIVA Program (BD) and ModFit LT software.
10
Cell Cycle Analysis and Clonogenic Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MTT and Flow Cytometry assays were performed as previously described47 (link)54 (link). For Flow Cytometry assay, MCF-7 cells transfected with different plasmids were stained with propidium iodide (PI) (BD Pharmingen, CA). Experimental data were collected by FACSCalibur (BD Biosciences, San Jose, CA, USA). Cell cycle profiles were determined using ModFit LT (BD Biosciences). For crystal violet staining assay, MCF-7 cells transfected with the appropriate plasmids were transferred to 35 mm plate, and were cultured until the recognizable clones appeared. Then, the cells were stained with crystal violet for 30 min at room temperature.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!