The siRNA-transfected cells were harvested 48 h after transfection. For the analysis, cells were harvested, fixed with ice-cold 70% ethanol overnight, and stored overnight at -4°C. The cells were stained with the Cell Cycle Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), and incubated for 30 min in the dark at room temperature, according to the manufacturer's protocol. The cells were analyzed using flow cytometry (FACSCalibur; BD Biosciences, San Diego, CA, USA). ModFit LT™ V3.3 (Verity Software House, Inc., Topsham, ME, USA) was used to analyze the results. A total of three independent experiments were performed.
Modfitlt v3
Manufactured by Verity Software House
Sourced in United States
ModFitLT V3.0 is a software application designed for flow cytometry data analysis. It provides tools for tasks such as cell population identification, histogram analysis, and statistical reporting. The software is intended for use in research and clinical laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (8 mentions)
Facscalibur flow cytometer, by BD (5 mentions)
Propidium iodide, by Merck Group (4 mentions)
Facscanto 2, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Cell cycle detection kit, by Keygen Biotech (3 mentions)
Facscanto flow cytometer, by BD (3 mentions)
Facscalibur system, by BD (3 mentions)
Facscan, by BD (3 mentions)
Pharm lyse lysing buffer, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit 2, by BD (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Lonza (1 mentions)
Novocyte 3000 flow cytometer, by Agilent Technologies (1 mentions)
Cellquest v3, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Nucleocounter nc 200, by ChemoMetec (1 mentions)
Dxflex flow cytometry, by Beckman Coulter (1 mentions)
Cell cycle kit, by Keygen Biotech (1 mentions)
54 protocols using modfitlt v3
1
Cell Cycle Analysis of siRNA-Transfected Cells
2
Cell Cycle and Apoptosis Analysis
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To analyze the cell cycle, cells were suspended in PBS buffer at 48 h post-transfection, fixed in 75% ethanol at 4°C overnight and stained with propidium iodide (PI) at room temperature for 30 min. For the analysis of cell apoptosis, harvested cells were suspended in PBS and stained using an Annexin V-fluorescein isothiocyanate/PI kit (cat. no. 556570, FITC Annexin V Apoptosis Detection kit II, BD Biosciences). Following staining, cells were immediately detected using a flow cytometer (FACSCalibur; BD Biosciences), and analyzed with ModFit LT™ V3.3 (Verity Software House, Inc.) for cell cycle analysis and FlowJo 10 (FlowJo LLC) for apoptosis analysis (30 (link),31 (link)).
3
Cell Cycle Analysis via PI Staining
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Propidium iodide (PI) single staining: cells (1 × ) were trypsinized and resuspended to obtain single-cell suspensions at 24 h post-transfection. Detached cells were fixed overnight at 4 °C in 70% ethanol, and were then stained with propidium iodide (Cell Cycle Detection kit; KeyGen) and analysed with a FACScan flow cytometer (BD Biosciences, USA) and the ModFit LT v3.3 software (Verity Software House, USA).
4
Cell Cycle Analysis of U87-MG Cells
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U87-MG cells were seeded into 6 well plates at 20 × 104 cells per well, and then treated with FL3 for 48 h. Afterwards, cells were trypsinized and centrifuged to have a unique pellet. The cell pellet is washed with PBS 1X and directly fixed with ethanol 70% for 30 min at 4 °C. The cell pellet was washed with PBS 1X, centrifuged at 850 g, and then treated with 100 µg/ml of ribonuclease (Sigma Aldrich), to finally stain it with 200 µl of Propidium Iodide (PI) (life technologies, Sigma Aldrich) at a concentration of 50 µg/ml (prepared from a 5 mg/ml stock solution). After 30 min of incubation at room temperature in the dark, cells were examined using a flow cytometer (MACS Quant, Miltenyi Biotec GmbH, Bergisch Gladbach R. F. A). A minimum of 20 × 103 cells was acquired per sample and data were analyzed using ModFit LT V3.3 software (Verity Software House, Topsahm, Maine, USA, https://www.vsh.com/products/mflt/ ). The percentage of cells in G0/G1, S and G2/M was determined from DNA content histograms.
5
Cell Cycle and Apoptosis Analysis
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Following BIX-01294 treatment, Huh7 and HepG2 cells were trypsinized and washed briefly with PBS once to remove the residual serum and trypsin. For cell cycle analysis, cells were fixed in 1 ml ice-cold 70% ethanol overnight at −20°C. Following fixation, cells were pelleted at 800 × g at 4°C for 5 min, the supernatant was aspirated carefully so as not to lose the pellet. The pellets were briefly washed twice with ice-cold PBS. The cells were resuspended with 500 µl propidium iodide (PI)/RNase A solution (50 µg/ml PI, 100 µg/ml RNase A and 0.1% Triton X-100 in PBS). The cells were incubated for 20 min at 37°C. DNA content was measured using a FACSCalibur instrument (BD Biosciences, Franklin Lakes, NJ, USA), and Modfit LT v3.3 (Verity Software House, Inc., Topsham, ME, USA) was used to analyze cell cycle phase distribution. For Annexin V/PI double staining analysis, following treatment with BIX-01294 as described above, HCC cells were double-stained with Annexin V-FITC and PI and incubated at room temperature for 5 min in the dark. A total of 10×104 cells from each group were analyzed using a FACSCalibur instrument.
6
Cell Cycle Analysis of A375 Cells
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Cell cycle in A375 cells was analyzed by flow cytometry by assessing cell DNA content. Cells were harvested after gentle Accutase treatment and centrifuged at 250xg during 5 min. The cell pellet was re-suspended in D-PBS (Lonza, Portsmouth, NH) before fixation with ice-cold ethanol under agitation. Fixed cells were stored at 4°C for 24 h, then treated with RNAse (Sigma-Aldrich, Tres Cantos, Madrid, Spain), stained with PI (Sigma-Aldrich), and analyzed in a Gallios flow cytometer (Beckman Coulter, Brea, CA). Data were analyzed with Modfit LT v3.3 software (Verity Software House, Topsham, ME).
7
Cell Proliferation and Cell Cycle Analysis
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Cells were plated at 420 cells/mm2 and cultured for 72 h. The total cell number for each replicate for each line was counted. Cells were re‐plated at 420 cells/mm2, and the experiment was repeated every 72 h for 15 days. The fold increase in cell number over day 0 was calculated using the mean value of each technical replicate for each cell line at each independent time point. Ki67 and caspase‐3 fluorescence immunocytochemistry was carried out as described previously 36 using antibodies listed in supplementary material, Supplementary materials and methods. CellTrace™ Violet proliferation studies were carried out according to the manufacturer's instructions (Thermofisher). The proliferation control and experimental samples were acquired on a Novocyte 3000 Flow Cytometer (Acea Biosciences, San Diego, CA, USA). Data were analysed using ModFit LT v3.3 software (Verity Software House, Topsham, ME, USA). Cell cycle analysis was carried out on the platforms described above using 5 μm Draq5 nuclear stain (BioLegend, San Diego, CA, USA) (15 min incubation) and cells fixed in 4% paraformaldehyde (PFA).
8
Cell Cycle Analysis of LA-N-1 Cells
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The cell cycle profile of the treated LA-N-1 cells was analyzed by staining the DNA content in the cells. LA-N-1 cells (1.5 × 105 cells/dish) were seeded in a 60 mm dish and incubated overnight. Afterwards, the cells were synchronized with 0.5% heat-inactivated FBS overnight and incubated at 37 °C. After synchronization, the cells were treated with either solvent control (0.5% ethanol) or different concentrations of DHA or EPA for 48 hours. On the day of analysis, the cells were permeabilized with 70% ethanol at 4 °C for 30 min. Subsequently, the cells were treated with propidium iodide (PI) and analyzed by flow cytometry (FACSCanto™ flow cytometer; BD BioSciences, San Jose, CA, USA) for cell cycle distribution using the ModFit LT V3.0 software (Verity Software House, Topsham, Maine, USA).
9
Cell Cycle Analysis by Flow Cytometry
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Cell-cycle progression under different conditions was evaluated by flow cytometry. After treatment, cells were harvested by digestion with 0.05% trypsin, washed twice with ice-cold 1× phosphate-buffered saline (1x PBS), and then fixed overnight at −20°C in 70% ethanol. The next day, the cells were washed with 1x PBS and incubated with 50 μg/ml propidium iodide (PI) and 50 μg/ml RNase A in 1x PBS on ice for 30 min in the dark. Flow cytometry was performed on a FACSCalibur system (BD Biosciences, San Jose, CA, United States) with CELLQuest v3.3 software (BD Biosciences), and cell-cycle distribution was analyzed with ModFit LT v3.0 software (Verity Software House, Topsham, ME, United States).
10
LPA-induced Cell Cycle Progression
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OVCAR5 cells were synchronized with a serum-free medium for 24 h and then pretreated with DMSO (vehicle, 0.1%), Ki16425 (10 μM), BB94 (10 μM), AG1478 (250 nM), LY294002 (10 μM) or rapamycin (100 nM) for 30 min, stimulated with 10 μM LPA up to 4 h. Cell-cycle progression under different conditions was evaluated by flow cytometry. After the treatment, cells were harvested by digestion with 0.05% trypsin, washed three times with ice-cold 1×PBS, fixed with ice-cold 70% ethanol for 30 min on ice, and incubated with 50 μg/ml PI and 50 μg/ml RNase A in 1×PBS for 30 min in the dark. All FACS analyses were performed on a FACSCalibur system (BD Biosciences), and cell cycle distribution was analyzed with ModFit LT v3.0 software (Verity Software House, Topsham, ME, United States).
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