The effects of afatinib on the cell cycle distribution were assessed using a propidium iodide staining‐based assay with the CycleTEST PLUS DNA Reagent Kit and FACSCaliber (Becton Dickinson, Franklin Lakes, NJ, USA). Untreated cells and cells treated with 0.1 μM afatinib for 24 or 48 h were subjected to cell cycle analysis. Doublets, cell debris, and fixation artifacts were gated out, and cell cycle analysis was carried out with CellQuest version 3.1 (Becton Dickinson).
Cellquest version 3
Manufactured by BD
Sourced in United States
CellQuest version 3.3 is a software application designed for flow cytometry data acquisition and analysis. It provides core functionalities for managing experimental workflows, acquiring and visualizing data from flow cytometers, and performing basic data analysis tasks.
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Lab products found in correlation
Facscalibur, by BD (8 mentions)
Binding buffer, by BD (4 mentions)
Propidium iodide, by Cell Signaling Technology (3 mentions)
Cycletest plus dna reagent kit, by BD (2 mentions)
Facs caliber, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Bromodeoxyuridine (brdu), by BD (2 mentions)
Annexin 5 fitc, by Cell Signaling Technology (2 mentions)
Facs scan cytometer, by BD (1 mentions)
Alexa fluor 647, by Bio-Rad (1 mentions)
Facscan laser, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
7 aminoactinomycin d, by Keygen Biotech (1 mentions)
Facsort flow cytometer, by BD (1 mentions)
Facscalibur system, by BD (1 mentions)
32 protocols using cellquest version 3
1
Cell Cycle Analysis of Afatinib Treatment
2
Immunological Profiling of Sheep
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100 µl of blood in EDTA was collected from immunized and unimmunized group of sheep at 0 days before challenge (49 DPV) and at 3DPC (52 DPV), 7 DPC (56 DPV), 14 DPC (63 DPV) and 21 DPC (70 DPV). 10 µl of conjugated antibodies, mouse anti-ovine CD4: ALEXA FLUOR®647 (Neat-1/10) dilution) and mouse anti-ovine CD8: RPE (Serotec, Immunological Excellence, USA) were mixed and incubated at room temperature for 30 min. Cells were washed with PBS, lysed with RBC lysis buffer and fixed with conjugated antibodies. Fixed cell pellets were analyzed in FACS scan cytometer (Becton Dickinson, USA). Appropriate isotype controls, mouse IgG2a negative control: RPE (for CD8) and mouse IgG2a negative control: ALEXA FLUOR®647 (for CD4) (Serotec, Immunological Excellence, USA) were used to overcome background fluorescence, if any. Stained cells were acquired in FACS scan cytometer and analyzed using software CELLQuest version 3.1 (Becton Dickinson, USA) after subtraction of the corresponding isotype control. Ten thousand events were recorded from each sample. Mean percentage variation in peripheral blood was analyzed for lymphocyte subpopulation by RPE fluorescence at (FL-2) and ALEXA FLUOR®647 fluorescence at (FL-4).
3
Cell Cycle Analysis of VSMC by Flow Cytometry
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The quantification of cell cycle distribution was examined using a FACScan laser flow cytometer (Becton Dickinson, San Jose, CA, USA). The VSMC was treated with TNF-α (10 ng/mL) in the absence or presence various concentrations (0.2 and 0.5 mg/mL) of HLP for 24 h; collected, rinsed with PBS twice; fixed in 70% ethanol at –20 °C overnight; and then stained with propidium iodide (PI) solution (20 μg/mL of PI, 20 μg/mL of RNase A, and 0.1% Triton X-100; all chemicals from Sigma-Aldrich, St Louis, MO, USA) for 20 min in the dark at room temperature. Each phase of cell cycle was presented as the cell number versus the DNA content as indicated by the intensity of fluorescence, and gated into subG1, G0/G1, S, and G2/M phases with CELLQuest Version 3.3 software (Becton Dickinson, San Jose, CA, USA).
4
Apoptosis and Cell Cycle Analysis
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SGC7901, SGC7901/5HRE-CEAp-RASSF1A and SGC7901/NC cells were seeded into 60-mm culture dishes and the hypoxia group was treated with CoCl2 (300 μm l−1) for 12 h. For apoptotic analysis, a FITC Annexin V Apoptosis Detection Kit I (Becton Dickinson, Franklin Lakes, NJ, USA) was used to analyze apoptosis according to the manufacturer's instructions. For cell cycle analysis, duplicate cells were fixed overnight with 75% ethanol at −20 °C, incubated with RNase A at 37 °C for 30 min and then incubated with propidium iodide at room temperature for 30 min. Cells were examined by flow cytometry and the data were analyzed by CellQuest version 3.3 software (Becton Dickinson).
5
Yeast Cell Cycle Synchronization Protocol
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Yeast cultures were inoculated from overnight cultures, grown using standard growth conditions and media. All cultures were grown in YPD-media containing glucose (2%) as carbon source at 28 °C, except for the temperature-sensitive strains (smc6-56 and top2-4) that were grown at 25 °C. For cell cycle synchronization, logarithmic cells grown at 28 °C were arrested in G1 using 3–5 μg/ml of alpha factor for 2–3 h. G2/M arrest was performed with 10–20 μg/ml of nocodazole for 2–3 h. Synchronization was verified microscopically and by flow cytometry (FACS) analysis (see Supplementary Fig. 3c ). FACS sample acquisition and analysis was performed using the BD CellQuest Version 3.3. Hydroxyurea (HU) was used at the concentration of 0.2 M unless otherwise indicated. For drug sensitivity and fitness viability assays, cells from overnight cultures were counted and diluted before being spotted in serial dilutions on YPD plates containing the indicated concentrations of HU and incubated at 28 or 30 °C as indicated for 2–3 days.
6
Quantification of Cellular ROS Levels
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After incubation under previously described cell culture conditions, ROS was detected by 5 μM DCFDA staining at 37 °C for 30 min. The image of cells was taken by a laser confocal scanning microscope (Leica Microsystems). ROS intensity was verified using an automated high-speed cytometer sorting system with BD Cell Quest version 3.3. Relative ROS production was measured using a fluorescence microplate reader (Bio Tek). Fluorescent was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
7
Annexin V Apoptosis Assay for HepG2 Cells
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In order to determine the effects of CDDP on HepG2 cell apoptosis, annexin V/propidium iodide (PI) staining was performed and analysis was conducted by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA), following the instructions of the annexin V FITC apoptosis detection kit (BD Biosciences). Briefly, cells were harvested, washed twice with precooled PBS and resuspended in 500 μl binding buffer following treatment with different concentrations of CDDP and NAC. Then, the cells were stained with 5 μl annexin V-FITC and 5 μl PI for 15 min at room temperature in the dark. The cell suspension was analyzed by flow cytometry. A blue (488 nm) excitation laser was used for both FITC and PI emission channels. The measured results were analyzed using CellQuest version 3.3 software (BD Biosciences).
8
Apoptosis and Cell Cycle Analysis
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All experiments were performed in triplicate. A total of 1×106 cells were plated in 6-well plates. Subsequent to 24 h treatment with the chemotherapy drugs (1 µM taxane, 500 nM TSA and 40 nM PS-341, individually or in combination) or DMSO as control, the cells were washed twice with PBS, stained with 5 µl Annexin V- Phycoerythrin (PE) and 5 µl 7-amino-actinomycin D (5 µg/ml) in 1X binding buffer following the protocol of the manufacturer (Nanjing Keygen Biotech, Co., Ltd, Nanjing, China) and analyzed using fluorescence-activated cell sorting (FACS) analysis. With regard to cell cycle detection, the cells were harvested and washed twice prior to being fixed in ice-cold 70% ethanol and stored at −20°C overnight. The cells were then washed once in PBS and resuspended in a solution of 5 mg/ml propidium iodide and 0.5 mg/ml RNase A (Thermo Fisher Scientific, Inc.) in PBS for 30 min in the dark prior to being sorted by FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). The results were analyzed with Cell Quest version 3.3 software (BD Biosciences).
9
Quantifying Apoptosis in HEK293/DsRed Cells
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The DsRed fluorescence intensity and apoptosis of HEK293/DsRed cells was evaluated using flow cytometry on a FACSort™ flow cytometer (BD Biosciences). The cells were harvested using 0.2% trypsin, washed twice with PBS and resuspended in PBS at a density of 1×106 cells. For analysis of apoptosis, the cells were fixed in 80% ice-cold ethanol overnight at −20°C and then incubated in 500 ml PBS containing 50 g/ml propidium iodide and 20 µg/ml RNase for 30 min. The data were analyzed using CellQuest version 3.3 software (BD Biosciences). The experiments were repeated three times.
10
Peripheral Blood T and NK Cells
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The T-and natural killer (NK)-cell profiles in the peripheral blood were evaluated throughout the preregistration period. The blood cell counts and differentials were measured using an automated blood cell count analyzer, and the lymphocyte fraction was determined by flow cytometry using forward scatter versus side scatter gating. The average number of lymphocyte subsets was calculated across the 5 data points. Immunophenotypic examination was performed using 2-or 3-color flow cytometry with a FACS-Calibur system running CellQuest, version 3.3, software (BD Biosciences, Franklin Lakes, NJ). Flow cytometry was performed at a single central laboratory (BML Inc). All antibodies used in the study were purchased from BD Biosciences. The lymphocyte subsets detected by flow cytometry were as follows: 13 The CD4/8 ratio was determined by 2wo-color analysis of CD4 and CD8 cell populations.
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