Briefly, 7 × 104 cells were seeded per well in 12-well plates and treated with 0.06–0.5 μM palbociclib or 0.5% (v/v) DMSO on the following day for 24 h. All floating and attached cells were harvested and fixed in 70% (v/v) ethanol for 16 h at –20°C. Prior to analysis, fixed cells were pelleted and washed in cold phosphate buffered saline, followed by staining with 10 μg/mL propidium iodide solution containing 20 μg/mL RNase for 30 min at 21°C in the dark. Stained cells were analyzed by BD FACSCanto IITM flow cytometer (BD Biosciences, MA, USA) with 10, 000 events collected for each reading. The distribution of DNA in different phases was determined using the ModFit software (Verity Software House, USA). The percentage of cells in each phase was calculated from three independent experiments.
Facscanto iitm flow cytometer
Manufactured by BD
Sourced in United States
The BD FACSCanto IITM is a flow cytometer designed for the analysis of cells and other biological particles. It is capable of detecting and measuring multiple parameters of cells, including size, granularity, and the expression of specific markers. The instrument uses a laser-based system to interrogate and analyze the samples.
Automatically generated - may contain errors
Lab products found in correlation
Modfit software, by Verity Software House (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Cell cycle assay kit, by Beyotime (1 mentions)
Modfit lt 3, by Verity Software House (1 mentions)
Cd3 fitc cd8 pe cd45 percp cd4 apc, by BD (1 mentions)
Facsdiva software version 6, by BD (1 mentions)
Xe 5000, by Sysmex (1 mentions)
7 protocols using facscanto iitm flow cytometer
1
Cell Cycle Analysis of Palbociclib Treatment
2
Flow Cytometry Protocol for Immunophenotyping
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Sample acquisition was performed in a BD FACSCanto IITM flow cytometer calibrated according to the EuroFlow Standard Operating Procedures [57 (link)]. External quality control assessment was achieved by participating in the EuroFlow Quality Assurance program [58 (link)]. A median number of 443,388 events was recorded per tube and stored as FCM standard (.fcs) 3.0 files, for subsequent analysis.
3
Measuring Intracellular ROS in MSCs
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MSCs treated with EP and control MSCs were trypsinized, washed, and
resuspended in 250 µL of DPBS and 5 µL of 2’-7’-dichlorodihydrofluorescein
diacetate 10 µM (H2DCF-DA) (Sigma-Aldrich) for 30 min at 37°C, in the dark
and under hypoxic conditions.23 (link) The fluorescence
intensity of 2’,7’-dichlorofluorescein (DCF) was measured in a BD FACS
CantoIITM flow cytometer. Median DCF fluorescence intensity
values, directly related to intracellular levels of ROS, were analyzed with
InfinicytTM Software (Cytognos).
resuspended in 250 µL of DPBS and 5 µL of 2’-7’-dichlorodihydrofluorescein
diacetate 10 µM (H2DCF-DA) (Sigma-Aldrich) for 30 min at 37°C, in the dark
and under hypoxic conditions.23 (link) The fluorescence
intensity of 2’,7’-dichlorofluorescein (DCF) was measured in a BD FACS
CantoIITM flow cytometer. Median DCF fluorescence intensity
values, directly related to intracellular levels of ROS, were analyzed with
InfinicytTM Software (Cytognos).
4
Cell Cycle Analysis in HCT116 and HCT15
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HCT116 and HCT15 cells were fixed in 70% cold ethanol for 24 h and treated with the Cell Cycle Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. A BD FACSCanto IITM flow cytometer (Lake Franklin, NJ, USA) was used to analyze the cell cycle.
5
Cell Cycle Analysis of Cancer Cell Lines
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24 h after seeding in culture flasks (75 cm2, 5 × 105 cells per flask) UD-SCC-5 and SAS cells were subjected to a combined treatment regimen as described above. 48 h after treatment cells were detached by trypsinization, washed twice with ice-cold PBS containing 2% FCS and fixed/permeabilized with 70% methanol on ice for 60 min. Finally, cells were washed twice with PBS and resuspended in PBS containing 1 μg/ml DAPI 15 min prior to analysis. 1 × 105 DAPI stained cells of every sample were analyzed using a BD FACSCanto-IITM Flow Cytometer (BD Biosciences, San Jose, USA). DNA histograms were plotted on a linear scale and cell cycle fractions, i.e. percentages of cells in G0/G1-, S- and G2/M-phase, were quantified using the ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA) upon cell doublet, aggregate, and debris discrimination via pulse processing.
6
Cytotoxicity Assay of RCC Cell Lines
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For cytotoxicity assessments, human RCC cell lines Caki-1 (metastatic, clear cell subtype, Von Hippel–Lindau (VHL) gene wild-type, well-differentiated) and ACHN (metastatic, papillary subtype, VHL gene wild-type, poorly differentiated) (4 × 104 cells/well) were incubated overnight in 48-well plates. Isolated RCC infiltrating Vγ9Vδ1 T cells or peripheral Vγ9Vδ2 T cells (5 × 103~1 × 104 cells/well) were added to target the tumor cells per well with and without activation. RCC-infiltrating Vγ9Vδ1 T cells were stimulated with anti-human Vδ1 Ab. For the cytotoxicity assay with Vγ9Vδ2 T cells, RCC cell lines were treated with hydroxymethylbutenyl-4-diphosphate (HMB-PP) for 20 h prior to performing cytotoxicity assays. After incubation for 24 h at 37 °C, tumor cell death was measured by eBioscienceTM Fixable Viability Dye eFluorTM 780 (Thermo Fisher Scientific, Waltham, MA USA) staining as per manufacturer’s instructions and analyzed by flow cytometry (BD FACS CantoIITM flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software software (BD Biosciences, San Diego, CA, USA).
7
Multicolor Flow Cytometry for Lymphocyte Enumeration
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For the dual-platform approach, the leukocyte count was obtained from a Sysmex XE-5000 device (Sysmex, Kobe, Japan) using light scattering technology. Leukocyte differentiation was done automatically by the instrument whenever possible. In case a sample was flagged by the instrument and differentiation was not possible, the sample was differentiated manually by an experienced technologist. The system was calibrated and quality controlled according to the manufacturer's instructions.
A stain/lyse/wash procedure was used for immunophenotypic analysis. Based on the physicians request, the following multicolor reagent combinations were used: CD3-FITC/CD8-PE/CD45-PerCP/ CD4-APC and/or CD3-FITC/CD16.56-PE/CD45-PerCP/CD19-APC (BD Biosciences). Samples were measured on a BD FACSCanto-II TM flow cytometer (BD Biosciences, San Jose, CA, USA) by analyzing 10,000 events within the lymphocyte gate. Acquisition and analysis was done by using the BD FACSDiva Software version 6 (BD Biosciences, San Jose, CA, USA). Gating was done manually by experienced lab technicians and verified by a medical supervisor. The following lymphocyte populations were enumerated: CD3+, CD3+/CD4+/CD8-, CD3+/CD4-/CD8+, CD3-/CD19+ and CD3-/CD56CD16+.
A stain/lyse/wash procedure was used for immunophenotypic analysis. Based on the physicians request, the following multicolor reagent combinations were used: CD3-FITC/CD8-PE/CD45-PerCP/ CD4-APC and/or CD3-FITC/CD16.56-PE/CD45-PerCP/CD19-APC (BD Biosciences). Samples were measured on a BD FACSCanto-II TM flow cytometer (BD Biosciences, San Jose, CA, USA) by analyzing 10,000 events within the lymphocyte gate. Acquisition and analysis was done by using the BD FACSDiva Software version 6 (BD Biosciences, San Jose, CA, USA). Gating was done manually by experienced lab technicians and verified by a medical supervisor. The following lymphocyte populations were enumerated: CD3+, CD3+/CD4+/CD8-, CD3+/CD4-/CD8+, CD3-/CD19+ and CD3-/CD56CD16+.
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