The cell cycle status was analysed with flow cytometry, based on quantification of DNA content, as described previously [21 (link)]. Next, A375 and RPMI7951 melanoma cells were treated with 1,25(OH)2D3 at 100 nM concentration, CPL304110 or AZD4547 at 5 µM concentration or with a combination of vitamin D with the selected FGFR inhibitor for 24 or 48 h. Trypsinized human malignant melanoma cells, as well as cells from the culture medium, were fixed together in 70% ethanol for 24–48 h at 4 °C. Following treatment with ribonuclease, the DNA was stained with propidium iodide (PI; Sigma Aldrich; Merck KGaA) for 30 min at 37 °C. The fluorescence of the PI-stained cells was measured by flow cytometry (FACSCalibur™; Becton, Dickinson and Company, Franklin, Lakes, NJ, USA). The results were analysed using the CellQuest™ Pro Software version 6.0 (Becton, Dickinson and Company) and expressed as a percentage of cells with DNA content corresponding to apoptotic/necrotic cells (subG1 fraction) or cells in G1, S and G2/M phases of the cycle.
Cellquest pro software version 6
Manufactured by BD
Sourced in United States
CellQuest Pro software version 6.0 is a flow cytometry data acquisition and analysis software. It provides a user interface for controlling flow cytometers and analyzing the collected data.
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Lab products found in correlation
Facscalibur, by BD (8 mentions)
Facscalibur flow cytometer, by BD (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti vegfr2, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Trypsin, by BD (1 mentions)
Trypsin inhibitor, by BD (1 mentions)
Rnase a, by BD (1 mentions)
Cycletest plus dna reagent kit, by BD (1 mentions)
Modfit lt software version 4, by Verity Software House (1 mentions)
Celltiter blue cell viability assay, by Promega (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Percp cy5.5 conjugated anti cd45 antibody, by BioLegend (1 mentions)
Counting beads, by Thermo Fisher Scientific (1 mentions)
F4 80 clone cl a3 1, by Bio-Rad (1 mentions)
Annexinv fitc from the annexin 5 fitc apoptosis detection kit, by Merck Group (1 mentions)
Trypsin, by Solarbio (1 mentions)
Facs lysing solution, by BD (1 mentions)
15 protocols using cellquest pro software version 6
1
Melanoma Cell Cycle Analysis by Flow Cytometry
2
Anti-VEGFR2 Expression in Melanoma Cells
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A375 melanoma cells were treated for 24 h with vitamin D compounds (calcitriol or calcipotriol) at 100 nM concentration, followed by 24 h incubation with cediranib at 500 or 1,000 nM concentration. Trypsinized human malignant melanoma cells at 1 × 106 density were harvested by centrifugation and rinsed two times in 3 ml of incubation buffer (0.5% bovine serum albumin in PBS). Following 10 min blocking in the incubation buffer, cells were stained for 30 min at room temperature with primary antibody anti-VEGFR2 (Cell Signaling, cat. no. 2479, rabbit monoclonal, 1:200) dissolved in the incubation buffer. Following rinsing 2× in incubation buffer, cells were incubated for 30 min with the secondary antibody (goat anti-rabbit IgG ThermoFisher Scientific A11008, 1:500) diluted in the incubation buffer. Cells were rinsed 2× with incubation buffer, dissolved in 0.5 ml of PBS and analyzed cytometrically on FACSCalibur™ (Becton, Dickinson and Company, Franklin, Lakes, NJ, USA) using the CellQuest™ Pro Software version 6.0 (Becton, Dickinson and Company). The results were expressed as a fluorescence geometric mean.
3
Melanoma Cell Cycle Analysis with Vitamin D and Cediranib
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The cell cycle status was analyzed based on quantification of DNA content using flow cytometry. Melanoma cells were treated for 24 h with vitamin D compounds (calcitriol or calcipotriol) at 100 nM concentration, followed by 72 h incubation with cediranib at 500 or 1,000 nM concentration. Trypsinized human malignant melanoma cells together with cells from culture medium were fixed in 70% ethanol for 24–48 h at 4°C, then treated with ribonuclease to remove any contaminating RNA, and the DNA was stained with propidium iodide (PI; Sigma–Aldrich; Merck KGaA) for 30 min at 37°C. The fluorescence of the PI–stained cells was measured by flow cytometry (FACSCalibur™; Becton, Dickinson and Company, Franklin, Lakes, NJ, USA). The results were analyzed using the CellQuest™ Pro Software version 6.0 (Becton, Dickinson and Company) and expressed as a percentage of cells with DNA content corresponding to apoptotic/necrotic cells (subG1 fraction) or cells in G1, S, and G2/M phases of the cycle. Supplementary Figure 1
4
PBMC Isolation and Viability Determination
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PBMCs were isolated by gradient density centrifugation (Lymphoprep solution, Nycomed Pharma; 600 x g, 20 min at 4˚C) (21 (link)). PBMCs were collected and washed (400 x g, 10 min at 4˚C) in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), re-suspended in tissue culture medium [RPMI-1640 supplemented with L-glutamate (2 mM), penicillin (1x105 IU/l), streptomycin sulphate (0.05 g/l), and 10% FBS; Gibco; Thermo Fisher Scientific, Inc.], and counted. The cell viability exceeded 98% as measured using propidium iodide (PI; 0.5 µg/ml/1x106 cells; 5 min at 22-25˚C; MerckMillipore) and a FACSCalibur flow cytometer with CellQuest Pro software version 6.0 (both from Becton Dickinson).
5
Antiapoptotic Effect Measurement in Tissues
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Livers and spleens were perfused with phosphate buffer saline (PBS), then removed, cut into small pieces, and stored in a deep freezer at −20 °C until use. The antiapoptotic effect was measured using the Annexin V-FITC Apoptosis Detection Kit (eBioscience, San Diego, CA, US) following the manufacturer's instructions. Flow cytometric analysis was performed on a BD FACSCalibur using Cell Questpro software version 6.0 (BD Biosciences).
6
Annexin V-Fluos RBC Phosphatidylserine Labeling
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After treatment, two million RBCs were resuspended in 25 μL of Ringer and incubated for 45 minutes at room temperature in the dark with Fluos–labeled annexin V (1:25, Roche, Indianapolis, IN) to label exposed PS. After washing, the RBCs were analyzed with a flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes NJ, USA) and Cellquest Pro software version 6.0 (BD Biosciences, Franklin Lakes NJ, USA) was used. Data analysis was performed with Cyan Summit software v4.3 (Beckman-Coulter, Fullerton CA, USA). Results are expressed as percentages of annexin V–positive RBCs.
7
GnRH-Induced Cell Cycle Analysis
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The HK1 cells were seeded into 100-mm culture plates at a density of 4.0×105 cells/dish in 6 ml culture medium. The cells were treated with GnRH at a concentration of 10-9 M for 48 h. Untreated cells were used as a control. The cells were prepared for cell-cycle analysis using a CycleTEST PLUS DNA Reagent kit (BD BioSciences, San Jose, CA, USA). A pellet containing 5×105 cells was gently resuspended in 250 μl solution A, containing trypsin (BD Biosciences), followed by 200 μl solution B, containing trypsin inhibitor and RNase A (BD Biosciences), and incubated at room temperature for 10 min each. A total of 200 μl propidium iodide (PI) was added and the cell suspensions were incubated at 4°C in the dark for 10 min. Flow-cytometric analysis of the cellular DNA content was performed using Cell Quest Pro software version 6.0 (BD Biosciences) on a FACS Calibur flow cytometer (BD BioSciences) and the results were analysed using ModFit LT™ software version 4.0 (Verity Software House, Inc., Topsham, ME, USA).
8
Cell Cycle Synchronization and Analysis
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Treatment with double-thymidine or nocodazole (Noc; Sigma-Aldrich; Merck KGaA) was conducted as described previously 38 (link). Cells were synchronized to the G2 phase via incubation with 5 µM RO-3306 for 16 h at 37 °C as described in a previous report 6 (link).
For cell cycle analysis, cells were harvested, washed, fixed and stained as described previously 30 . Cell cycle quantification was performed using a FACS Calibur instrument and CellQuest Pro software, Version 6.0 (both BD Biosciences).
The activity of caspase-3/7 was determined using a Caspase-Glo®3/7 assay (Promega Corporation), according to the manufacturer's instructions. Cell viability and proliferation assays were conducted using the Cell Titer-Blue® Cell Viability assay (Promega Corporation, cat. no. G808B), as described previously 30 .
For cell cycle analysis, cells were harvested, washed, fixed and stained as described previously 30 . Cell cycle quantification was performed using a FACS Calibur instrument and CellQuest Pro software, Version 6.0 (both BD Biosciences).
The activity of caspase-3/7 was determined using a Caspase-Glo®3/7 assay (Promega Corporation), according to the manufacturer's instructions. Cell viability and proliferation assays were conducted using the Cell Titer-Blue® Cell Viability assay (Promega Corporation, cat. no. G808B), as described previously 30 .
9
Annexin V-FITC Apoptosis Assay
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Apoptosis was determined using an fluorescein isothiocyanate (FITC) Annexin V apoptosis DTEC KIT (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. First, A549 cells were incubated with sinomenine for 24 and 48 h. The cells were collected and washed twice with cold PBS and then resuspended in 1X Binding Buffer at a concentration of 1×106 cells/ml. Subsequently, 100 µl of the suspension (1×105 cells) was transferred to a 5 ml culture tube, and 5 µl FITC-conjugated Annexin V with 10 µl propidium iodide (PI) were added. The cells were gently vortexed and incubated for 15 min at 25°C in the dark. Finally, 400 µl of 1X Binding Buffer was added to each tube and analyzed using the FACSCalibur™ flow cytometer and CellQuest Pro software version 6.0 (BD Biosciences) within 1 h.
10
Apoptosis Assay for Breast Cancer Cells
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MCF-7 and MDA-MB-231 breast cancer cells (3 × 105 cells/well) were seeded in a six-well plate and were incubated at 37 °C in a 5% CO2 atmosphere. At 24 h post incubation, cultured cells were treated with either free TC or TC-SF-NPS (25 μg/ml) and further incubated for 24 h. The cells were harvested by trypsinization and rinsed trice with PBS. Subsequently, cells were stained by 5 µl Annexin V-FITC for 10 min and 10 µl PI for 5 min, respectively, in the dark at room temperature, according to manufacturer’s protocol (ThermoFisher Scientific, Waltham, MA). Analysis of cell apoptosis was carried out using flow cytometry (BD FACSCaliburTM; BD Biosciences, San Jose, CA). Data collection and analysis is done with CellQuest Pro software version 6.0 (BD Biosciences, San Jose, CA). The results are expressed as the rate of apoptosis (the percentage of early + late apoptotic cells) (Al Saqr et al., 2021a ,b ).
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