The cell cycle was assayed with Vybrant DyeCycle Orange Stain (Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA). The cells were seeded into 12-well plates at an initial density of 0.150 × 106 cells/mL in complete medium. After 72 h of incubation with or without 100 μM of adenine nucleotides or adenosine, the cells were washed with PBS and resuspended in 1 mL of RPMI 1640 medium with 10% FBS at a density of 106 cells/mL. The cells were stained according to the manufacturer’s instructions and analyzed by flow cytometry. The histograms were analyzed with the ModFit LT 4.1 software (Verity Software House, Topsham, ME, USA). The results are presented as the percentage of cells in particular phases of the cycle (G0G1, S, and G2M).
Modfit lt 4
Manufactured by Verity Software House
Sourced in United States
ModFit LT 4.0 is a software application for analyzing flow cytometry data. It provides tools for DNA cell cycle and proliferation analysis. The software is designed to be user-friendly and offers a range of features to help researchers analyze their data effectively.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (6 mentions)
Accuri c6, by BD (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Cfda se, by Thermo Fisher Scientific (4 mentions)
Proliferation wizard module, by Verity Software House (4 mentions)
Pi rnase staining buffer, by BD (4 mentions)
Facscalibur, by BD (3 mentions)
Cycletest plus dna kit, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Facsaria 2 flow cytometer, by BD (3 mentions)
Rnase a, by Takara Bio (3 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Flow cytometry, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Facscanto 2 cytometer, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Facscanto a, by BD (2 mentions)
Cell cycle kit with pi staining, by BD (2 mentions)
Annexin 5 fitc and pi, by BD (2 mentions)
Cycletest plus dna reagent kit, by BD (2 mentions)
74 protocols using modfit lt 4
1
Cell Cycle Analysis with Adenine Nucleotides
2
Cell Cycle Analysis of Preadipocytes
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Flow cytometry experiments were performed to analyze the cell cycle distribution in proliferating and differentiating preadipocytes after Dnmt3a3 overexpression or negative control using the Cell Cycle and Apoptosis Analysis kit (Beyotime, Jiangsu, China) according to the manufacturer’s protocols. The cells were cultured in 12-well plates (19,000 cells per well) in normal growth medium (as described above) for 48 h after transfection or in growth medium for 6 h after transfection and then transferred to adipogenic medium (as described above) for 48 h. At 80% confluency, the cells were transfected with pSDS-Dnmt3a3 or pSDS using Lipofectamine 3000 (Invitrogen). After transfection for 48 or 54 h, the cells were detached with trypsin, washed once with phosphate buffered saline (PBS), and fixed in 66% ethanol at 4°C. Following 24 h fixation, the cells were washed with PBS and stained with 20 µg/ml propidium iodide (PI) in PBS containing 20 µg/ml RNase A and incubated for 30 min at 37°C in the dark. Next, CytoFLEX flow cytometer (Beckman Coulter, Brea, United States) was used to quantify the DNA content in each sample. A total of 10,000 events were acquired for each sample, and the cell-cycle distribution was analyzed using ModFit LT 4.1 software (Verity Software House, Topsham, United States).
3
Cell Cycle Analysis by Flow Cytometry
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The cells were digested to single cells and centrifuged at 1,000 rpm for 5 min to collect cell precipitates. Then 70% ethanol was added and cells were fixed overnight at 4°C. After washing with PBS, the cells were resuspended in 500 µl PBS containing 50 µg/ml PI, 100 µg/ml RNase A, and 0.2% Triton X-100 and incubated for 30 min at room temperature. Flow cytometry (CytomicsFC500; Beckman Coulter) was used to detect 20,000-30,000 cells. The results were analyzed by cell cycle fitting software ModFit LT 4.1 (Verity Software House).
4
Cell Cycle Analysis by Flow Cytometry
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Flow cytometry was performed to analyze cell cycle distribution. Cells were harvested, washed with cold PBS and fixed in cold 70% ethanol overnight at 4°C, followed by treatment with 0.25 mg/ml RNase A for 30 min at room temperature. After staining with propidium iodide (PI; 0.05 mg/ml; Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, the samples were analyzed using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and was analyzed using ModFit LT 4.1 (Verity Software House, Topsham, ME, USA).
5
Cell Cycle Analysis by Flow Cytometry
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Each cell line was seeded in a 12-well plate exposing in drugs for 72 h. Then the cells were collected, centrifuged, washed with ice-cold PBS and resuspended with 70 % alcohol. After fixed in -20 °C for 12 h, the cells were washed and stained with PI and ribonuclease A (Beytime Instituted of Biotechnology, China) at room temperature for 30 min. Cell cycle data were assessed by BD LSRFortes flow cytometry (SanJose, CA, USA) and analyzed by Modfit LT 4.1 (Verity Software House, Topsham, USA).
6
Cell Cycle Analysis of HCC Cells
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HCCLM3 cells were seeded in 6-well plates (2.5×105/well). At 24 h, cells were treated with 200 µmol/l polydatin for an additional 48 h at 37°C. Following trypsinization, cells were washed with PBS and fixed with 75% ethanol for 4 h at 4°C. Cells were washed twice with PBS and covered with 0.5 ml PBS containing 20 µl RNaseA and PI for 30 min at 4°C. The cell cycle progression of HCC cells was detected using the FACSCalibur flow cytometer with an excitation wavelength of 488 nm, and emission wavelength of at 670 nm. Cell cycle progression was analyzed using ModFit LT 4.1 (Verity Software House, Topsham, ME, USA) by gating live cells.
7
Apoptosis and Cell Cycle Analysis by Flow Cytometry
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Cell apoptosis was analyzed using an Annexin V/PI kit (BD Biosciences) and flow cytometry. DU145 cells were collected by trypsinization, washed twice in PBS, fixed with 500 µl binding buffer, and incubated with 5 µl Annexin V-EGFP and the DNA binding dye PI (50 mg/ml) for 5–15 min at 37°C in the dark. Finally, the cells were analyzed using an Elite flow cytometer (CytoFLEX S; Beckman Coulter, Inc.) with a peak fluorescence gate to distinguish aggregates after 1 h. Next, the cell cycle distribution was assessed. Briefly, following treatment with 100 µM DIDS for 48 h, the cells were collected by trypsinization, washed in PBS and fixed in 70% ethanol for 30 min at 4°C. After being washed with PBS, the cells were incubated with PI (50 mg/ml) and RNase (1.0 mg/ml) for 30 min at 37°C in the dark. Finally, the cells were washed, and red fluorescence was analyzed using an Elite flow cytometer (CytoFLEX S, Beckman Coulter, Inc.) with a peak fluorescence gate to distinguish aggregates. All data were analyzed by ModFit LT 4.1 (Verity Software House, Inc.).
8
Cell Cycle Analysis of Transduced Cells
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MGC80-3 and AGS cells transduced with shCon or shPYCR1 were trypsinized, centrifuged at 2500 g for 5 min, rinsed with cold phosphate-buffered saline (PBS), and fixed with 75% ethanol. The cells were subsequently washed with PBS and incubated with 5 μL of RNase (200 U/mL, DNase-free) for 5 min. Then, 10 μg/mL propidium iodide (Sigma) was added. Cell cycle status was evaluated on a FACS Calibur instrument (BD Biosciences, San Jose, CA) in triplicate, and the analysis was facilitated using the Modfit LT 4.1 (Verity Software House, Topsham, ME).
9
Cell Cycle Analysis of Reversine and Analogs
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Cell cycle analysis was performed on at least 30,000 events for each sample with FACSCantoTM A using BD Diva Software 6.1.3 (BD Bioscience, San Jose, CA, USA) and the DNA profile was analyzed by ModFit LT 4.1 (Verity Software House, Topsham, ME, USA). Cells were treated with 0.1% DMSO, 1 µM Reversine or different concentrations of each 1 –3 (1 µM, 2.5 µM, 5 µM and 10 µM) for 24 h, detached with trypsin-EDTA, collected by centrifugation and washed three times with PBS. Cells were stained with propidium iodide using BD CycletestTM Plus DNA Kit (BD Bioscience) and then analyzed by flow cytometry. Results obtained are the merge of three experiments.
10
Cell Cycle Analysis using BD Reagents
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BD cell cycle reagent (BD Biosciences) was used according to the manufacturer's protocol. ESCC cells were washed with cold PBS and then fixed in 70% ethanol. After fixation, the cells were stained with propidium iodide. Finally, the cell suspensions were detected and analysed with a flow cytometer (BD Biosciences) and evaluated with ModFit LT 4.1 software (Verity Software House).
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