For the in vitro tumor-cell challenge, 5–10×105 tumor-infiltrating lymphocytes (TIL), which were isolated from tumor tissues or lymphoid organs, were cultured with OVA peptide (1 µl/ml) for 4–5 hours in the presence of 1 µl/ml of Brefadin A (Sigma), washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to block nonspecific binding, and then stained with CD8-PE-Cy5 and IFNγ-FITC or control antibodies according to the manufacturer’s instructions (BD Pharmingen). Tumor antigen-specific CD8 T cells were identified by staining with OVA-tetramer (Beckman Coulter) and TRP-2 pentamer (ProImmune). Cells were analyzed using FACScan flow cytometer and FlowJo version X.10 (Tree Star, Ashland, OR) software.
Facscan flow cytometer
Manufactured by Tree Star
Sourced in United States
The FACScan flow cytometer is an analytical instrument designed to quantify and characterize cells and particles based on their physical and fluorescent properties. It utilizes the principles of flow cytometry to rapidly analyze multiple parameters of individual cells or particles in a sample.
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Lab products found in correlation
Ifn γ fitc, by BD (1 mentions)
Cd8 pe cy5, by BD (1 mentions)
Flow cytometry, by Merck Group (1 mentions)
Annexin 5 fitc staining kit, by BD (1 mentions)
Annexin 5 fitc kit, by Bio-Techne (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Beyotime (1 mentions)
Cd4 percp, by BioLegend (1 mentions)
Igd pe, by BD (1 mentions)
Flow jo software version 7, by Tree Star (1 mentions)
Cd20 efluor 450, by Thermo Fisher Scientific (1 mentions)
Cd38 pe cy7, by BD (1 mentions)
Cd19 apc, by BD (1 mentions)
Cd3 fitc, by BD (1 mentions)
Hla dr fitc, by BD (1 mentions)
Annexin 5 pe 7 aad detection kit, by Yeasen (1 mentions)
10 protocols using facscan flow cytometer
1
Tumor-Infiltrating Lymphocyte Characterization
2
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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During cell cycle analyses, cells in the logarithmic growth phase were washed three times with PBS. Suspended cells at a concentration of 1 × 106/ml were fixed with 70% ethanol for 0.5 h. Following propidium iodide (PI) staining, DNA content was measured using flow cytometry (BD Biosciences, San Jose, CA, USA).
The cellular apoptosis assay was performed using the PI/Annexin V-APC Apoptosis Kit (Sigma) according to the manufacturer's instructions. A FACScan flow cytometer and FlowJo software (Tree Star Inc., Ashland, OR) were used to analyze the staining data. All experiments were repeated in triplicate.
The cellular apoptosis assay was performed using the PI/Annexin V-APC Apoptosis Kit (Sigma) according to the manufacturer's instructions. A FACScan flow cytometer and FlowJo software (Tree Star Inc., Ashland, OR) were used to analyze the staining data. All experiments were repeated in triplicate.
3
Cell Cycle Analysis of Saos-2 Cells
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Saos-2 cells were counted using flow cytometry and then seeded onto culture plates (6 wells, 1 × 105 cells per well) for analysis of the cell cycle. The incubation medium was changed after 24 h to DMEM containing 1% FBS, which rendered the cells quiescent for 24 h. Saos-2 cells were then subjected to a number of treatments and after 24 h the cells were gently fixed by adding 80% ethanol, and then stored in a freezer for 2 h. Next, the cells were placed in an ice bath and exposed for 5 min to 0.25% triton X-100 before being resuspended in 300 mL of PBS solution that contained 0.1 mg/mL of RNase and 40 mg/mL of propidium iodide. The cells were then incubated for 20 min at room temperature in a darkroom before analysis of the cell cycle with a FACScan flow cytometer and FlowJo software, ver. 7.1.0 (Tree Star, US). At least 10,000 cells were counted for each measurement.
4
Analyzing Sub-G1 Cell Population
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The sub-G1 cell population was analyzed by flow cytometry using propidium iodide (PI) staining, as described by Zhao et al.32 (link) Briefly, the single cell suspensions were adjusted at a density of 1×105 cells/well (CSC-CM of U2OS-SC and MG6-SC) and were plated into 6-well plates. The cells were treated with or without the aforementioned agents for 72 h. PI staining was performed for DNA analysis using a FACScan flow cytometer and the FlowJo software, ver. 7.1.0 (Tree Star, USA).
5
Apoptosis Quantification by Flow Cytometry
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The percentage of apoptosis cells were detected by flow cytometry using the Annexin V-FITC staining kit (BD Pharmingen, USA). Briefly, after transfected with siRNA for 24 h, cells were digested, collected and washed three times with PBS, and then resuspended in binding reagent. Annexin v-FITC and PI were then added according to the manufacturer's instructions. A FACS can flow cytometer and FlowJo software (Tree Star Inc., OR, USA) were utilized to analyze the data.
6
Cell Viability Assay using Annexin V-FITC and PI
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The cell viability was determined by flow cytometry using the Annexin V-FITC kit (Trevigen, Gaithersburg, MD, USA). MCF-7 and T-84 cells were seeded at a high density (2 × 105 cells/cm2) in 6-well plates. After 24 h, the cells were induced with PTS and PTSO for 48 h at the IC50 concentration for each cell line. The cells were detached with the TrypLE Express Enzyme (ThermoFisher Scientific, Waltham, MA, USA), washed with PBS, and collected by centrifugation at 300× g for 10 min. Then, the cells were washed again and incubated with annexin-V FITC and propidium iodide (PI) in an annexin-V binding buffer for 15 min. After incubation, the cells were diluted with the binding buffer and examined immediately in a FACScan flow cytometer, using FlowJo (v.7.6.5, Tree Star, Inc., Ashland, OR, USA). This assay was performed in duplicate.
7
Annexin V-FITC and PI Staining
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Approximately 4 × 103 of SW480 and LS1034 cells were gathered and washed by PBS (Beyotime) twice. Afterward, following the manufacturer’s instructions 10 μL Annexin V-FITC (Carlsbad) and 5 μL PI staining solution (Carlsbad) were added to SW480 and LS1034 cells and kept in the dark at room temperature for 15 min. The Flow cytometry results were analyzed by FACScan flow cytometer (Treestar, Inc., San Carlos, CA, USA).
8
Multiparameter Flow Cytometry Analysis
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Expression of the following markers on CD4 T cells and CD19 B cells was assessed after 4 days in culture using the following monoclonal antibodies (mAb): CD4-PerCP (Biolegend, San Diego, CA, USA), CD19-APC (BD, Erembodegem-Aalst, Belgium), CD25-PE (DAKO, Glostrup, Denmark), and HLA-DR-FITC (BD). For analysis of B cell differentiation the following monoclonal antibodies were used: CD3-FITC (BD Pharmingen, San Diego, CA, USA), CD19-APC-Alexa Fluor750 (BD), CD20-eFluor450 (eBioscience, San Diego, CA, USA) CD27-APC (ImmunoTools GmbH, Friesoythe, Germany), CD38-PE-CY7 (BD), and IgD-PE (BD). IgG1-FITC/IgG1-PE (Immunotech, Marseille, France) was used as isotype control.
Ki67 intracellular staining was performed using mouse-anti-human Ki67-FITC staining set (eBioscience) following manufactures instructions. Cell acquisition was done using a FACScan flow cytometer and data were analysed with FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).
Ki67 intracellular staining was performed using mouse-anti-human Ki67-FITC staining set (eBioscience) following manufactures instructions. Cell acquisition was done using a FACScan flow cytometer and data were analysed with FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).
9
Cell Cycle and Apoptosis Analysis
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After transfection, cells in the logarithmic growth phase were washed three times with PBS. Suspended cells at a concentration of 1 * 106/ml were fixed with 70% ethanol at 4 °C overnight. Following propidium iodide (PI) staining, DNA content was measured using flow cytometry (BD Biosciences, San Jose, CA, USA). The cellular apoptosis assay was performed by using the PI/Annexin V-FITC Apoptosis Kit (Sigma, USA) according to the manufacturer's instructions. A FACS can flow cytometer and FlowJo software (Tree Star Inc., Ashland, OR) were used to analyze the staining data.
10
Apoptosis Assay Using Annexin V-PE/7-AAD
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Cell apoptosis was detected using an Annexin V-PE/7-AAD Detection Kit (Yeasen, Shanghai, China) according to the instructions of the manufacturer. Briefly, after incubation with MPA (10 μM) or DMSO (control) for 48 h, cells were trypsinized, washed, and resuspended in binding buffer. Next, 5 μl of Annexin V-PE and 10 μl of 7-AAD were added to the cell suspension and incubated in the dark at 4°C for 15 min. A FACScan flow cytometer and FlowJo software (Tree Star Inc., Ashland, OR, USA) were used to analyze the cells.
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