For fluorescence-activated cell sorting (FACS) analysis, LNCaP cells (2.0 × 105 for each experimental point) were left untreated or treated for the indicated times with Bisphenol A (10, 50 or 100 μM). Cells were collected, resuspended in 400 μl of a buffer containing 0.1% NP-40, 0.1% sodium citrate, 1 mg/ml RNAse A, 50 μg/ml propidium iodide and 0.1 mM EDTA. Cells were then incubated in the dark for 1h and samples were analyzed by a fluorescence-activated cell sorting (FACS) Calibur flow cytometer using Cell Quest software (Becton Dickinson, BD Biosciences). Results from three independent experiments were analyzed using Cell Quest software (Becton Dickinson) and ModFit LT version 3 Software (Verity, Topsham, ME, USA).
Cellquest software
Manufactured by BD
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CellQuest software is a data acquisition and analysis software designed for flow cytometry applications. It provides tools for acquiring, processing, and analyzing flow cytometry data.
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Lab products found in correlation
Facscalibur, by BD (1124 mentions)
Facscalibur flow cytometer, by BD (1010 mentions)
Facscan, by BD (344 mentions)
Facscan flow cytometer, by BD (249 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (155 mentions)
Propidium iodide, by Merck Group (124 mentions)
Facscalibur cytometer, by BD (117 mentions)
Cycletest plus dna reagent kit, by BD (80 mentions)
Facscalibur system, by BD (77 mentions)
Rnase a, by Merck Group (73 mentions)
Annexin 5 fitc, by BD (62 mentions)
Flow cytometry, by BD (53 mentions)
Propidium iodide, by BD (48 mentions)
Facscanto 2, by BD (43 mentions)
Facs caliber flow cytometer, by BD (40 mentions)
Facscalibur flow cytometry, by BD (38 mentions)
Ionomycin, by Merck Group (37 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (36 mentions)
Facscalibur instrument, by BD (34 mentions)
Facsort flow cytometer, by BD (30 mentions)
5 116 protocols using cellquest software
1
FACS Analysis of Bisphenol A Effects
2
Cell Cycle and Differentiation Analysis
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Cell-cycle profile was analyzed using flow cytometry. Briefly, 2 × 106 cells were washed with PBS, fixed in 80% ethanol and resuspended in 50μg/ml propidium iodide (Sigma) and 125U/ml RNase A (Sigma). For cell cycle profile, DNA content was analyzed by flow cytometry using FACScan and Cell Quest Software (Becton Dickinson, USA). For detection of cell differentiation antigen cd11b and cd114, 1 × 106 cells were washed twice with PBS, incubated with PE-conjugated cd11b or cd114 antibody or PE-conjugated IgG isotype control antibody at 4°C for 30 min and analysed by flow cytometry using FACScan and Cell Quest Software (Becton Dickinson, USA) [12 (link), 13 (link), 29 (link)].
3
Epicutaneous Challenge-Induced Immune Response
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In the epicutaneous challenge-mice, the lymph nodes were collected, cells were isolated and re-stimulated for 16 h at 37 °C, 5% CO2 with a cell stimulation cocktail containing PMA at 20 ng/mL and ionomycin at 1 µM in the presence of brefeldin A and monesin at 10 µg/mL. Subsequently, after washing and fixation, cells as CD4pos T cells were assessed for intracellular content of IFN-γ, IL-17A, IL-4, IL-5 by flow cytometer using a FACSCalibur flow cytometer equipped with CellQuest software (BD Biosciences) and were analyzed using CellQuest software (Becton-Dickinson, San Jose, CA). The skin tissue removed was immediately fixed in 10% buffered formalin. The tissue was then processed and embedded in paraffin. Five-micrometer tissue sections were prepared and stained using H&E and toluidine blue methods. The stained paraffin-embedded sections were examined and qualitatively evaluated using light photomicroscopy for inflammatory cell influx. All slides were examined with light microscopy at a magnification of 10x or 40× (Axio Imager A1; Carl Zeiss) calibrated with a reference micrometer slide. The quantification of the number of cells in a section of 400 mm2 per slide was done using Axio Vision Rel 4.8 software.
4
Quantifying Neuronal Apoptosis by Flow Cytometry
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APC-conjugated annexin-V and 7-amino-actinomycin D (7-AAD) (Becton Dickinson Biosciences, BDB, San Jose, CA, USA) were used to determine quantitatively the percentage of apoptotic neurons by flow cytometry. Cells were stained with annexin V-APC and 7-AAD, following the manufacturer’s instructions, and were analysed on a FACScalibur flow cytometer (15 mW argon ion laser tuned at 488 nm; CellQuest software, Becton Dickinson Biosciences) using the CellQuest software (BDB). Both GFP+ and GFP− cells were analyzed separately, and the annexin V-APC-stained cells that were 7-AAD-negative were considered to be apoptotic (see, also, Supplementary Fig. S3 for the flow cytometry workflow for apoptosis).
5
Characterizing Dental Pulp and Bone Marrow Stem Cells
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Dental pulp stem cells and BMMSCs were characterized using flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA). Flow cytometry was done on BD FACSCalibur cytometer and data were processed with CellQuest software (Becton Dickinson Biosciences). A total of 0.5 × 106 DPSCs and BMMSCs were incubated with specific individual monoclonal antibodies, conjugated with fluorescence isothiocyanate (FITC), phycoerythrin (PE) in 250 μl phosphate buffered saline for 30 min in the dark at room temperature. The following cell surface antigens were observed CD29-FITC, CD90-FITC, CD34-FITC, CD105-PE, and CD45-peridium chlorophyll protein complex (BD Pharmingen). CD 29 and CD 90 are known stromal precursor of BM and highly expressed in MSCs.[22 (link)23 (link)] CD105 (endoglin) is expressed in MSCs and hematopoietic stem cells. CD 34 and CD 45 are cell surface markers widely used in isolation and identification of hematopoietic stem and progenitor cells. They are expressed exclusively on hematopoietic stem cells.[24 ] Mouse isotype-matched IgG served as a negative control (BD Pharmingen). 100,000 labeled cells were acquired and analyzed using CellQuest software [Figure 1 (Becton Dickinson)].
6
Quantifying Apoptosis and Intracellular VEGF
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For determination of apoptotic cell death, cells were stained with propidium iodide (PI) and Annexin-V-FITC (BD Pharmingen, CA) and analyzed on flow cytometer (FACS Callibur, BD Biosciences, CA). Electronic compensation of the instrument was done to exclude overlapping of the emission spectra. Total 10,000 events were acquired for analysis using CellQuest software (BD Biosciences, CA) [26] (link). Annexin-V-positive cells were regarded as early apoptotic cells [24] (link). For quantification of intracellular VEGF, cells pre-treated with Brefelidin A were harvested, washed with PBS, fixed with 4% para-formaldehyde for 10 min and permeabilized with 0.1% Triton-X-100 for 5 min and incubated with anti-VEGF antibody followed by TRITC-conjugated secondary antibody. Fluorescence was determined flow cytometrically using CellQuest software (Becton Dickinson, San Jose, CA).
7
Lung Immune Cell Profiling in Mice
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After the last challenge with VTn, mice were killed by the injection of lethal dose of sodium pentobarbital anesthesia and the tracheas were cannulated. The airway lumina was washed with 4 × 0.5 mL of Hank’s balanced salt solution (HBSS, Gibco BRL, Grand Island, NY) + 10 mM ethylenediaminetetraacetic acid (EDTA). The resulting BAL fluids were immediately centrifuged at 800 rpm, 4 °C, for 10 min. The supernatant was removed and kept at −20 °C. Three sections of lung were dissociated into single cell suspensions by mechanical disruption in Gentle MACS dissociator (Miltenyi). Cells were isolated and restimulated for 16 h at 37 °C, 5% CO2 with a cell stimulation cocktail containing PMA at 20 ng/mL and ionomycin at 1 µM in the presence of brefeldin A and monesin at 10 µg/mL. Subsequently, after washing and fixation, cells as CD4pos T cells were assessed for intracellular content of IFN-γ, IL-17A, IL-4, IL-5 by flow cytometer using a FACSCalibur flow cytometer equipped with CellQuest software (BD Biosciences) and were analyzed using CellQuest software (Becton-Dickinson, San Jose, CA).
8
Cell Cycle and Apoptosis Analysis of MM Cells
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For cell cycle analysis, MM cells were collected using cold phosphate buffer saline (PBS) and then immobilized using 70% cold ethanol solution overnight. Prior to propidium iodide (PI; Solarbio, Beijing, China) staining, RNase was used to remove RNA in the samples. The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur (Becton–Dickinson, San Jose, CA, USA) and analyzed using Cell Quest software (Becton–Dickinson).
For apoptosis analysis, after transfection for 48 h, MM cells were collected with PBS, and then these cells were suspended in binding buffer. Annexin V-combined fluorescein isothiocyanate (Annexin V-FITC; Solarbio) and PI (Solarbio) were added to the reaction mixture, and MM cells were simultaneously incubated with Annexin V-FITC and PI at 37 °C for 15 min in a dark room. The apoptotic MM cells were identified by FACSCalibur (Becton–Dickinson) and analyzed using Cell Quest software (Becton–Dickinson).
For apoptosis analysis, after transfection for 48 h, MM cells were collected with PBS, and then these cells were suspended in binding buffer. Annexin V-combined fluorescein isothiocyanate (Annexin V-FITC; Solarbio) and PI (Solarbio) were added to the reaction mixture, and MM cells were simultaneously incubated with Annexin V-FITC and PI at 37 °C for 15 min in a dark room. The apoptotic MM cells were identified by FACSCalibur (Becton–Dickinson) and analyzed using Cell Quest software (Becton–Dickinson).
9
Quantifying PGP Expression in GBM Cell Lines
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The expression of PGP in the different GBM cell lines U87, U118 and GBM11 was assessed by flow cytometry using monoclonal antibodies labelled with fluorochromes. For each assay, 106 cells were used and data on at least 10,000 events were collected using a FACSCalibur flow cytometer, CellQuest software (Becton-Dickinson, Franklin Lakes, NJ, USA), and analysed using CellQuest™ software (Becton-Dickinson). Since PGP is a membrane protein, cells were centrifuged at 300 × g and incubated for 15 min at room temperature with the monoclonal antibodies: anti-human PGP [fluorescein isothiocyanate (FITC) and mouse anti-human P-glycoprotein (BD Biosciences)].
10
Apoptosis and Cell Cycle Analysis
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Cell apoptosis was determined using an annexin V–FITC apoptosis detection kit (KeyGen Biotechnology, Nanjing, China) according to the manufacturer's instructions. Briefly, A549 cells were treated with PepE (0.5, 2.0, or 4.0 μM) or 5‐Aza‐dC (2.0 μM) for 48 h. Cells were harvested by trypsinization, washed twice with pre‐chilled PBS, and centrifuged at 671 × g for 5 min. The cell pellet was resuspended in binding buffer and stained with annexin V–FITC/propidium iodide. After incubation at 15°C for 15 min in the dark, the samples were quantified by Becton Dickinson FACSCalibur flow cytometry (Franklin Lakes, NJ, USA), and data were analyzed using CellQuest software (Becton Dickinson).
Cell cycle analysis was undertaken using a cell cycle detection kit (KeyGen Biotechnology) according to the manufacturer's instructions. Briefly, A549 cells were treated with PepE (2.0 μM) or 5‐Aza‐dC (2.0 μM) for 48 h. The cells were harvested and fixed with 70% alcohol at 4°C for 12 h, DNA was stained with propidium iodide in the presence of 1% DNase‐free RNase A at 37°C for 30 min before flow cytometry analysis (FACSCalibur; Becton Dickinson). The distribution of cells in distinct cell cycle phases was analyzed using CellQuest software (Becton Dickinson).
Cell cycle analysis was undertaken using a cell cycle detection kit (KeyGen Biotechnology) according to the manufacturer's instructions. Briefly, A549 cells were treated with PepE (2.0 μM) or 5‐Aza‐dC (2.0 μM) for 48 h. The cells were harvested and fixed with 70% alcohol at 4°C for 12 h, DNA was stained with propidium iodide in the presence of 1% DNase‐free RNase A at 37°C for 30 min before flow cytometry analysis (FACSCalibur; Becton Dickinson). The distribution of cells in distinct cell cycle phases was analyzed using CellQuest software (Becton Dickinson).
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