Flow cytometry was used to evaluate the cell cycle profile, apoptosis, and mitochondrial membrane potential of FKB-treated HeLa cells. The cell cycle profile of untreated controls and HeLa cells treated with FKB for 48 hours was evaluated using BD CycletestTM Plus DNA kit according to the manufacturer’s protocol. Briefly, harvested control and treated HeLa cells were incubated with 250 µL of Solution A (10 minutes), followed by 200 µL of trypsin inhibitor and RNase buffer Solution B (10 minutes), and finally by 200 µL of propidium iodide (PI; 10 minutes). The samples were then analyzed by BD FACSCalibur using BD CellQuest Pro software (BD, USA). For apoptosis quantification, BD Annexin-V/PI apoptosis kit (BD, USA) was used according to the manufacturer’s protocol. In brief, harvested control and FKB-treated HeLa cells were incubated with 5 µL of AnnexinV-FITC and 5 µL of PI for 15 minutes. Then, the samples were topped up with 400 µL of binding buffer and analyzed by BD FACSCalibur using BD CellQuest Pro software (BD, USA). BD MitoScreen JC-1 kit was used for quantification of mitochondrial membrane potential. In brief, harvested control and FKB-treated HeLa cells were incubated with 500 µL of JC-1 working solution (15 minutes), washed, and resuspended with 500 µL of assay buffer. The samples were then analyzed by BD FACS Calibur using BD CellQuest Pro software (BD, USA).
Cellquest pro software
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CellQuest Pro software is a flow cytometry data acquisition and analysis application developed by BD. It provides a user interface for controlling flow cytometry instruments and analyzing the resulting data.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (519 mentions)
Facscalibur, by BD (512 mentions)
Propidium iodide, by Merck Group (72 mentions)
Facscalibur cytometer, by BD (61 mentions)
Facscan flow cytometer, by BD (38 mentions)
Rnase a, by Merck Group (36 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (34 mentions)
Facscan, by BD (32 mentions)
Facscalibur system, by BD (23 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (18 mentions)
Propidium iodide, by Thermo Fisher Scientific (18 mentions)
Annexin 5 fitc, by BD (18 mentions)
Facs caliber, by BD (15 mentions)
Pe annexin 5 apoptosis detection kit, by BD (15 mentions)
Flow cytometry, by BD (14 mentions)
Modfit lt software, by Verity Software House (13 mentions)
Propidium iodide, by BD (13 mentions)
Facscalibur flow cytometry, by BD (13 mentions)
Facscanto 2, by BD (12 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
1 799 protocols using cellquest pro software
1
Flow Cytometric Analysis of FKB-Induced Cell Cycle, Apoptosis, and Mitochondrial Changes in HeLa Cells
2
Measuring ROS and Mitochondrial Membrane Potential
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ROS (reactive oxygen species) generation was measured by FACS using the oxidant-sensitive probe 2′,7′-dichlorodihydrofluorescein (H2DCF-DA). C. zeylanicum EO was administered at a dose of 10 µg/mL for 24 h. The treated cells were then harvested and incubated with 10 µM H2DCF-DA in HBSS with 0.1% BSA for 30 min at 37 °C. To generate ROS as a control, we used H2O2 at a concentration of 5 mM. The cells were then washed in HBSS with 0.1% BSA and analysed with a FACSCalibur cytofluorimeter and CellQuest Pro BD software (BD).
The mitochondrial membrane ΔΦ was measured by FACS using JC-1 staining [49 (link)]. After washing in PBS, the cells were incubated with 2.5 mg/mL JC-1 for 20 min at room temperature in the dark. After two washes in PBS, the samples were immediately analysed with a FACSCalibur cytofluorimeter and CellQuest Pro BD software (BD). As a control, we used a depolarised sample treated with the ionophore valinomycin for an additional 15 min after JC-1 staining.
The mitochondrial membrane ΔΦ was measured by FACS using JC-1 staining [49 (link)]. After washing in PBS, the cells were incubated with 2.5 mg/mL JC-1 for 20 min at room temperature in the dark. After two washes in PBS, the samples were immediately analysed with a FACSCalibur cytofluorimeter and CellQuest Pro BD software (BD). As a control, we used a depolarised sample treated with the ionophore valinomycin for an additional 15 min after JC-1 staining.
3
Phospho-STAT protein analysis in MG-63 cells
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MG-63 cells were incubated on discs for 72 h, washed with PBS and incubated with 10 ng/mL cytokine for 20 min at 37 °C, harvested, washed and fixed with 2% PFA for 30 min at RT and permeabilized with 0.1% TX-100 for 5 min at RT, as was previously described [21 (link)]. The MG cells cultured on wells without discs were used for comparison. Then the cells were stained o/n at 4 °C with Alexa Fluor 488-conjugated anti-phospho-STAT1, anti-phospho-STAT3, or anti-phospho-STAT6 antibodies, as is indicated in the figure legends and collected on an FACS Calibur cytometer with CellQuest Pro software (Becton Dickinson, Franklin Lakes, NJ, USA). Unstained cells or cells stained with isotype-matched IgG antibodies served as a negative control. All experiments were performed three times. The mean fluorescence intensities were quantitatively analyzed (20,000 events in each variant). The images were collected on a Becton Dickinson FACS Calibur using Cell Quest Pro Software.
4
Flow Cytometry Analysis of Oxidative Stress
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FACS Calibur flow cytometer (BD Bioscience) in conjunction Cell-Quest Pro software was used for flow cytometry. 20 μM of 2’,7’-dichlorofluorescin diacetate (Sigma) was added to the renal single cell suspension and incubated in dark for 15 min at 37°C. Fluorescence intensity was acquired at FL1 channel with BD FACS calibur (BD Biosciences). 10,000 events were taken and Cell Quest Pro software (Becton Dickinson, Franklin Lakes, New Jersey, USA) was used for analysis.
5
Measuring Homologous Recombination Capacity
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To measure HR capacity, PDCs were co-transfected with pDR-GFP + pCBASceI (a gift from Dr. Maria Jasin, Addgene #26475 and #26477) or pDR-GFP + pFUGW-RFP (transfection efficiency control) (1:1) for a total of 2.5 μg DNA using lipofectamine LTX with Plus Reagent (Invitrogen) according to the manufacturer’s protocol in OptiMEM (Gibco) in 6-well plates (Corning 3506). Six hours post-transfection, media was replaced with media containing vehicle or inhibitors. Cells were incubated for an additional 48 h, collected, and analyzed for GFP and RFP expression using a FACSCalibur cytometer with CellQuest Pro software (BD Biosciences).
To evaluate the effects of DDR inhibitors on HR capacity, PDCs were transfected with pDR-GFP and selected with 1–10 μg/ml puromycin 48 h later. Post-selection, PDR-GFP stable lines were seeded in 6-well plates, allowed to attach overnight, and transfected with pCBASceI or pFUGW-RFP using lipofectamine LTX as above. Six hours post-transfection, media was replaced with media containing vehicle or inhibitors. Cells were incubated for an additional 72 h, collected and analyzed for GFP expression using a FACSCalibur cytometer with CellQuest Pro software (BD Biosciences).
To evaluate the effects of DDR inhibitors on HR capacity, PDCs were transfected with pDR-GFP and selected with 1–10 μg/ml puromycin 48 h later. Post-selection, PDR-GFP stable lines were seeded in 6-well plates, allowed to attach overnight, and transfected with pCBASceI or pFUGW-RFP using lipofectamine LTX as above. Six hours post-transfection, media was replaced with media containing vehicle or inhibitors. Cells were incubated for an additional 72 h, collected and analyzed for GFP expression using a FACSCalibur cytometer with CellQuest Pro software (BD Biosciences).
6
Measuring Homologous Recombination in DR-GFP Cells
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HR was measured using DR-GFP cells as described previously (37 (link)). Briefly, DR-GFP U2OS cells were transfected with control siRNA, TOPORS siRNA, RAD51 siRNA, or TOPORS and RAD51 siRNAs using Lipofectamine RNAiMax (Invitrogen). For the rescue experiment, TOPORS-depleted DR-GFP U2OS cells were transfected with TOPORS-WT or TOPORS-T515A. At 12 h after transfection, cells were transfected with pCBA-I-SceI plasmid using the Turbofect transfection reagent (Thermo Fisher Scientific). After 48 h, cells were harvested and analyzed for GFP expression by flow cytometry (FACSCalibur, BD Biosciences). The data were analyzed using CellQuest Pro Software (BD Biosciences). For recovery of HR repair by reintroducing untagged RAD51-WT or RAD51-K57R/K70R, DR-GFP U2OS cells were transfected with control or RAD51 3′UTR siRNAs. At 12 h after transfection, RAD51-WT, RAD51-K57/70R and pCBA-I-SceI plasmids were transfected again into DR-GFP U2OS cells. GFP-positive cells were counted 48 h after transfection using flow cytometry (FACSCalibur). The data were analyzed by CellQuest Pro Software (BD Biosciences).
7
Arbutin Induces Apoptosis in MC3T3-E1 Cells
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MC3T3-E1 cells were seeded in six-well plates at a density of 2×105 cells/well. Following a 24-h incubation, cells were treated with arbutin at concentrations of 0, 10, 50 and 100 µM. Cells were harvested after 3 days at RT and fixed with 70% ethanol for 12 h at 4°C. Cells were washed three times with PBS and stained with propidium iodide (PI) staining solution (Beyotime Institute of Biotechnology, Haimen, China) for 30 min at 37°C in the dark. DNA content was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with CellQuest Pro software (Version 5.2; BD Biosciences) and ModFit LT software (Version 3.0; Verity Software House, Inc., Topsham, ME, USA). For apoptosis analysis, cells treated with arbutin were harvested and stained with fluorescein isothiocyanate-labeled Annexin-V and PI (Dojindo Molecular Technologies, Inc.) in the dark for 15 min at RT. The cell apoptotic rate was assessed using a FACSCalibur flow cytometer (BD Biosciences) with CellQuest Pro software (Version 5.2; BD Biosciences).
8
Cell Cycle Analysis of β-ELE Treated Cells
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T24 and 5637 cells were seeded into 6-well plates at density of 5×104 cells/well and cultured at 37°C with 5% CO2 for 24 h. Cells were treated with different concentrations of β-ELE (0, 25, 50 and 75 µg/ml) at 37°C for 24 h, washed twice with PBS and fixed in cold 70% (v/v) ethanol for 48 h at 4°C. Prior to analysis, cells were washed with PBS, incubated with 100 µl RNase A (0.1 mg/ml) for 30 min at 37°C, stained with 2 µl propidium iodide (PI; Sigma-Aldrich; Merck KGaA) and incubated for another 30 min at RT in the dark according to the manufacturer's instructions. Samples were analyzed by flow cytometry using CellQuest™ Pro software (version 5.1 BD CellQuest Pro Software, BD Biosciences).
9
Apoptosis Induction in T24 Cells
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T24 cells were seeded into 6-well plates at density of 5×104 cells/well and cultured at 37°C with 5% CO2 for 24 h. Cells were incubated with cisplatin (20 µM) and/or β-ELE (50 µg/ml) for 24 h at 37°C, harvested and washed with PBS, re-suspended in binding buffer and stained with Annexin V-FITC and PI (cat. no. C1062M; all Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The stained samples were analyzed by flow cytometry (BD Biosciences). Data were analyzed using CellQuest™ Pro software (version 5.1 BD CellQuest Pro Software, BD Biosciences).
10
Cell Cycle and Proliferation Assays
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RL95-2 (6 × 105) and HEC-1-A (3 × 105) cells were seeded into 6-well plates in their respective media. The next day, the indicated drugs were added in fresh medium, and the cells were incubated for an additional 24 or 48 h. For cell cycle analysis, the cells were fixed in 70% ice-cold ethanol and stored at −20 °C overnight. The fixed cells were then centrifuged (1000 rpm, 5 min), washed twice with ice-cold PBS supplemented with 1% FBS, and stained with PI solution (5 μg/mL PI in PBS, 0.5% Triton X-100, and 0.5 μg/mL RNase A) for 30 min at 37 °C in the dark. For each condition, 10,000 cells were analyzed using a BD FACScalibur™ flow cytometer and Cell Quest Pro software (version 5.1) (BD Biosciences, Franklin Lakes, NJ, USA).
For cell proliferation assays, following the incubation protocol described above, the cells were incubated for an additional 1 h with 10 μM BrdU (BD Pharmingen BrdU Flow Kit, San Diego, CA, USA). The medium was then discarded, and the cells were fixed at room temperature for 30 min and treated with FITC-conjugated anti-BrdU antibody (BD Pharmingen). After washing, the cells were incubated with 7-AAD and analyzed using a BD FACScalibur™ flow cytometer and CellQuest Pro software (BD Biosciences).
For cell proliferation assays, following the incubation protocol described above, the cells were incubated for an additional 1 h with 10 μM BrdU (BD Pharmingen BrdU Flow Kit, San Diego, CA, USA). The medium was then discarded, and the cells were fixed at room temperature for 30 min and treated with FITC-conjugated anti-BrdU antibody (BD Pharmingen). After washing, the cells were incubated with 7-AAD and analyzed using a BD FACScalibur™ flow cytometer and CellQuest Pro software (BD Biosciences).
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