Tumor tissue was digested in a 0.3% collagenase/0.1% hyaluronidase solution, pressed through a nylon mesh filter to obtain a single cell suspension and incubated in red cell lysis buffer (0.17 M Tris-HCl, 0.16 M NH4Cl) for 3 min, spun down, and resuspended in FACS buffer (PBS + 1.5% FBS). Equal numbers of cells were stained with a viability dye and combinations of the following antibodies: CD8a-PECy7 (eBioscience, San Diego, CA, USA, 25-0081-82), F4/80-PECy7(eBioscience, 25-4801-82), CD206-FITC (Biolegend, 141704), Ly6G-APC (eBioscience, 17-5931-81), CD11b-PE (eBioscience, 12-0112-82), CD45-PE-Cy5 (eBioscience, 15-0451-83), IFN-γ-APC (eBioscience, 17-7311-82), and SIINFEKL pentamer-PE (ProImmune, Oxford, UK). For autophagy analysis using Cyto-ID staining of bone marrow derived macrophages, cells were first stained for surface markers (CD80-PE-Cy5 (eBioscience, 15-0801-81) and CD206-APC (eBioscience, 17-2061-82)) followed by staining with Cyto-ID for 30 min at 37 °C, per the manufacturer’s protocol (Enzo Life Sciences, Farmingdale, NY, USA, ENZ-51031). Flow cytometric data were acquired on a BD FACSCanto II cytometer and analyzed using FACSDiva software version 9.2 (BD Biosciences, San Jose, CA, USA).
Facs diva software version 9
Manufactured by BD
Sourced in United States
FACS Diva software version 9.0 is a flow cytometry data analysis software. It provides tools for acquisition, analysis, and management of flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Facs celesta sorp flow cytometer, by BD (3 mentions)
Facs celesta sorp instrument, by BD (2 mentions)
Cd206 apc, by Thermo Fisher Scientific (1 mentions)
Ly6g apc, by Thermo Fisher Scientific (1 mentions)
Cd11b pe, by Thermo Fisher Scientific (1 mentions)
Ifn γ apc, by Thermo Fisher Scientific (1 mentions)
Cd8a pecy7, by Thermo Fisher Scientific (1 mentions)
Cd45 pe cy5, by Thermo Fisher Scientific (1 mentions)
Cd80 pe cy5, by Thermo Fisher Scientific (1 mentions)
F4 80 pe cy7, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Cd206 fitc, by BioLegend (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
Tryple reagent, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Cxcr3 bv421, by BD (1 mentions)
Ccr7 bv711, by BD (1 mentions)
Ccr6 bb515, by BD (1 mentions)
7 protocols using facs diva software version 9
1
Flow Cytometry Analysis of Tumor Immune Cells
2
Apoptosis Assay of HCT116 and HT-29 Cells
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In total, 6 × 105 HCT116 or 7 × 105 HT-29 cells were seeded in 60 mm plates and incubated overnight at 37 °C under 5% CO2. The next day, cells were treated with Qc at the concentrations indicated above for 24 h. At the end of the treatment, cells were harvested, including dying cells floating in the medium, washed with PBS, and processed with the FITC Annexin V Apoptosis Detection Kit I (556547, Becton Dickinson—BD, San Jose, CA, USA). Cell death was evaluated by BD FACS Celesta and data acquired with FACS Diva software, version 9.2 (BD).
3
Flow Cytometric Analysis of HLA-I Expression
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iRhom2KO HTB94 cells and its WT counterparts were grown in 6–well plates and then treated with 10 µg/ml brefeldin A (Sigma-Aldrich, US) or monensin (BD GolgiStop™ Protein Transport Inhibitor containing monensin, BD Biosciences, US) for 3 h. Then, cells were harvested with TryPLE reagent (Gibco, part of Thermo Fisher Scientific) and processed for flow cytometry analysis. Briefly, HTB94 were stained with FITC anti-HLA Class I [W6/32], isotype IgG2a (Abcam, Cambridge, UK) or PE anti-HLA Class I [W6/32], isotype IgG2a (BioLegend, San Diego, CA) at room temperature for 15 min according to the manufacturer’s instruction, washed with PBS and then resuspended in 300 µl PBS. Cells were analysed with FACS Celesta SORP flow cytometer and FACS Diva software version 9.0 (BD Biosciences, CA, US).
4
Profiling Ascites Immune Cells
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Ascites immune cells from 4 patients were profiled using a panel of markers to enumerate monocytes/macrophages (CD14+), neutrophils (CD15+), and lymphocytes cells (CD45+). We further phenotyped ascites using a panel of markers that distinguish lymphocyte cell subsets (T cells CD3+, T helper CD4+, T cytotoxic CD8+, B cells CD19+, and NK cells CD16+-56+) using a BD FACS Celesta SORP instrument (Table 3 ). Analyses were completed using FACS Celesta SORP flow cytometer and FACS Diva software version 9.0 (BD Biosciences, San Jose, CA, USA).
5
Flow Cytometry Analysis of Lymphocytes
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FCM assay was conducted on the stained cells with a FACSCanto II (BD Biosciences) flow cytometer equipped by FACSDiva Software version 9.0 (BD Biosciences). At least 40,000 lymphocytes per sample were recorded. Analysis of FCM raw data was performed by FlowJo_V10 software (BD Biosciences).
6
Evaluation of CD4 T-cell Differentiation
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To evaluate CD4 T-naïve differentiation, the lymphocyte component of the ascites was analyzed in all culture conditions from 3 patients. Specifically, after 72 h of co-culture with hA-MSCs, the maturation status (naïve, central memory, CM; effector memory, EM; terminal effector memory, TEM) of CD4+ cells was analyzed and compared to the control lymphocytes. We phenotyped T cells using a panel of markers that distinguish lymphocyte cell subsets in T helper CD4+ Th1/Th2/Th17. In detail, we analyzed the CD4+ T cells’ differentiation states using different marker combinations, CD4+ naïve (CD45+CCR7+), CD4+ CM (CD45RA-CCR7+), CD4+ EM (CD45RA-CCR7-), and CD4+ TEM (CD45RA+CCR7-), and the lymphocytes cells subsets in T-helper CD4+ using specific marker combinations, Th1 (CD4+CXCR3+CCR4-), Th2 (CD4+CCR4+CCR6-), and Th17 (CD4+CCR4+CCR6+). The cells were labeled with CD45 APC-Cy7, CD3 BV510, CD4 BV786, CD45RA PE Cy-7, CCR7 BV711, CXCR3 BV421, CCR4 APC, and CCR6 BB515—all from BD Biosciences (BB: Brilliant Blue, BV: Brilliant Violet). Analyses were conducted using a BD FACS Celesta SORP instrument, FACS Celesta SORP flow cytometer, and FACS Diva software version 9.0 (BD Biosciences, CA, USA).
7
Immunophenotypic Analysis of Murine HSPCs
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The immunophenotypic characterization of isolated hematopoietic stem and progenitors cells (HSPC) from animals previously treated with 5-FU was performed by flow cytometry (FACSAria™ III, BD Biosciences, San Jose, CA, USA) using the BD Mouse Hematopoietic Stem and Progenitor Cell Isolation Kit (BD Biosciences, San Jose, CA, USA), with comprises the following antibodies: Sca-1-PE-Cy7, c-Kit-PE, CD34-FITC, and the Lineage-APC antibody cocktail (CD3, CD45R, Ly6C, Ly6G, CD11b and TER-119).
Isolated HSPCs were stained by incubation with the antibodies specific for HSPCs, hematopoietic lineages, and a viability dye: c-Kit, Sca-1, Lineage and the viability dye 7-Amino-Actinomycin D (7-AAD). The acquisition of at least 10,000 events were performed into a flow cytometry (FACSAria™ III, BD Biosciences, San Jose, CA, USA). The results were analyzed using FACSDIVA software version 9.0 (BD Biosciences, San Jose, CA, USA) and FlowJo version 10.6 (BD Biosciences, San Jose, CA, USA). The analyses used as a gate strategy, a first selection for singlets (FSC-H vs. FSC-A), followed by a second gate for viability (7-AAD unstained cells). Next, only viable cells were gated for granularity vs. lineage markers, and only lineage negative cells were analyzed for HSPC specific markers.
Isolated HSPCs were stained by incubation with the antibodies specific for HSPCs, hematopoietic lineages, and a viability dye: c-Kit, Sca-1, Lineage and the viability dye 7-Amino-Actinomycin D (7-AAD). The acquisition of at least 10,000 events were performed into a flow cytometry (FACSAria™ III, BD Biosciences, San Jose, CA, USA). The results were analyzed using FACSDIVA software version 9.0 (BD Biosciences, San Jose, CA, USA) and FlowJo version 10.6 (BD Biosciences, San Jose, CA, USA). The analyses used as a gate strategy, a first selection for singlets (FSC-H vs. FSC-A), followed by a second gate for viability (7-AAD unstained cells). Next, only viable cells were gated for granularity vs. lineage markers, and only lineage negative cells were analyzed for HSPC specific markers.
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