On day 22, the tumors were excised, dissected into small pieces using a scalpel, digested in collagenase digestion buffer (1% collagenase in RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.) for 2 h at 37°C with agitation and the cells in the resulting suspension were counted. Cells were passed through a 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) following disaggregation A total of 1×106 cells in 100 µl were stained. The cells were labeled using rat anti-mouse CD11b-allophycocyanine (1:50; cat no. 553312; BD Biosciences), hamster anti-mouse CD11c-fluorescein isothiocyanate (1:100; cat no. 553801; BD Biosciences), or rat anti-mouse F4/80-A488 (1:50; cat no. MCA497A488; Serotec, Bio-Rad Laboratories, Inc., Hercules, CA, USA) antibodies on ice for 30 min, followed by analysis by flow cytometry using FACSCalibur or LSR II flow cytometers (BD Biosciences); data were analyzed using CellQuest 6.0 software (BD Biosciences).
Cellquest 6
Manufactured by BD
Sourced in United States
CellQuest 6.0 is a software application for flow cytometry data acquisition and analysis. It provides a user interface for controlling flow cytometry instruments and performing basic data analysis functions.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (5 mentions)
Facscalibur, by BD (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
70 m cell strainer, by BD (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis kit, by BD (1 mentions)
Trypsinization, by Thermo Fisher Scientific (1 mentions)
Cd105, by Santa Cruz Biotechnology (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis detection kit, by Abcam (1 mentions)
Accuri c6, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Facscanto 2, by BD (1 mentions)
Rnase a, by 4A Biotech (1 mentions)
Annexin 5 fitc apoptosis detection kit, by 4A Biotech (1 mentions)
Cell cycle detection kit, by Keygen Biotech (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Sungene Biotech (1 mentions)
10 protocols using cellquest 6
1
Tumor Dissociation and Cell Analysis
2
Apoptosis Analysis of Cancer Cells
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The apoptosis of MGC-803 and BGC-823 cells following different treatments was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis kit (BD Biosciences) by flow cytometry. Cells were collected and stained with Annexin V-FITC and propidium iodide solutions. Subsequently, the apoptosis of the cells was examined using a FACSCalibur flow cytometer (BD Biosciences) with CellQuest 6.0 software (BD Biosciences, Franklin Lakes, NJ, USA).
3
Cell Surface Antigen Expression Analysis
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The expression levels of cell surface antigen were evaluated using flow cytometry. When cultures reach >80% confluency at 37°C, 5% CO2 and 95% humidity, adherent cells were removed from the tissue culture polystyrene flasks via trypsinization (Invitrogen; Thermo Fisher Scientific, Inc.) and washed twice with DMEM/F12. All cells were incubated with fluorescein isothiocyanate-conjugated mouse anti-rat monoclonal antibodies against rat CD45 (cat. no. 554877), CD73 (cat. no. 551123), CD90 (cat. no. 554894) all obtained from BD Biosciences), CD34 (cat. no. sc-7324; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and CD105 (cat. no. ab11414; Abcam, Cambridge, MA, USA) for 40 min at room temperature at a dilution of 1:50. Following antibody incubation, data were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest 6.0 software (BD Biosciences).
4
Annexin V-FITC/PI Apoptosis Assay
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Cell apoptosis was determined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BioVision, Inc.). In brief, HMEC-1 cells were collected and suspended in 200 µl binding buffer, and then double-stained with 5 µl Annexin V-FITC and 10 µl PI at room temperature for 20 min in the dark. Stained apoptotic cells (Annexin V+ and PI−) were identified by a flow cytometer (Accuri C6; BD Biosciences) and data were analyzed utilizing Cell Quest 6.0 software (BD Biosciences).
5
Cell Cycle and Apoptosis Analysis by FCM
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Flow cytometry (FCM) was used to analyze the cell cycle and apoptosis. The cell cycle was analyzed by ethanol-fixed cells stained with propidium iodide (PI) in buffer containing RNase A. The DNA content was assessed by using the FACS calibur flow cytometer (BD Biosciences). Cell apoptosis was detected using the annexin V–FITC apoptosis detection kit (KeyGEN BioTECH, Nanjing, China). Briefly, cells were collected and washed with PBS twice, and stained for 15 min at room temperature with annexin V-FITC and (PI). Following that, cell apoptosis was examined using FACS calibur. The percentage of apoptotic cells was calculated with CellQuest 6.0 (BD Biosciences).
6
Annexin V-FITC Apoptosis Assay
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To measure the extent of 786-O and ACHN cell apoptosis, an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was used, according to the manufacturer's instructions (eBioscience; Thermo Fisher Scientific, Inc.). At 36 h post-transfection, cells were harvested and centrifuged at 1,200 × g for 5 min at room temperature. Then cells were washed once with PBS, and suspended in 1X binding buffer. A total of 5 µl Annexin V-FITC was added to 100 µl of cell suspension. Cells were washed with 2 ml of binding buffer and suspended in 200 µl of 1X binding buffer. Finally, 200 µl cell suspensions were mixed with 5 µl propidium iodide (PI) staining solution then assessed by flow cytometry (BD FACSCantoII; BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Cellquest 6.0 (BD Biosciences).
7
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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As previously described,[15 (link)] flow cytometry (FCM) was used to analyze cell cycle and apoptosis. The cell cycle was analyzed by ethanol-fixed cells stained with propidium iodide (PI) in buffer containing RNase A (Cat: FXP0211, 4A Biotech Co., Ltd, Beijing, China). The DNA content was assessed with a FACS Calibur flow cytometer (BD Biosciences, USA). Cell apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (Cat: FXP022, 4A Biotech Co., Ltd, Beijing, China). The percentage of apoptotic cells were calculated with CellQuest 6.0 (BD Biosciences, USA).
8
Cell Cycle and Apoptosis Analysis
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Cells were collected 48 h after siRNA transfection for cell cycle and apoptosis detection. FCM was used to examine the cell cycle and apoptosis. For cell cycle analysis, cells were treated by using a cell cycle detection kit (KeyGENBiotech, Nanjing, China) according to the manufacturer's instructions. In brief, cells were fixed for 2 h with ethanol and stained with propidium iodide (PI) in buffer containing 10 μg RNase A. The cells were then kept in the dark at room temperature for 30 min. Cell cycle distribution was assessed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, USA). Cell apoptosis was detected using the annexin V–FITC apoptosis detection kit (4A Biotech Co, Ltd, Beijing, China, Cat: FXP022). Cells were collected, washed twice with PBS and stained with annexin V-FITC and PI in the dark at room temperature for 15 min. The percentage of apoptotic cells was calculated using CellQuest 6.0 (BD Biosciences).
9
Cell Cycle Analysis Using Flow Cytometry
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The cells were seeded on plates, and, on the following day, cells were starved with a serum-free medium overnight. Cells were treated with the test compound for 24 h and collected. The cells were then washed twice with PBS, fixed in 70% cold ethanol, and incubated at −20 °C for 4 h or overnight. The cells were washed with cold PBS, and the fixed cells were resuspended with RNase A (100 g/mL) and shaken for 30 min. The cells were stained with PI (50 g/mL) for 15 min at room temperature, and the cell cycle analysis was conducted using flow cytometry (FACSCalibur flow cytometer; BD Biosciences, San Jose, CA, USA). The fluorescence intensity was analyzed using the program CellQuest 6.0 (BD Biosciences, San Jose, CA, USA).
10
Apoptosis Assessment of VPA-Treated Cells
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The cells (4 × 10 5 per well) were seeded in a six-well plate overnight and treated with varying concentrations of VPA for 4 days. To measure the extent of cell apoptosis, an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was used according to the manufacturer's instructions (Sungene Biotech, Tianjin, China). In brief, the cells were harvested and suspended in 1 × binding buffer. Then, 100 µL of the cell suspension was incubated with 5 µL of Annexin V-FITC for 10 min, followed by incubation with 5 µL of PI solution for another 5 min. The labeled cells were then assessed by flow cytometry and analyzed using Cellquest 6.0 (BD Biosciences, NJ, USA) [15] .
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