The number of 1×105 cells were harvested from fresh human PBMCs and TILs that were stained with anti-CD3 (PerCP-Cy 5.5 or APC-conjugated BD Bioscience), anti-CD8 (PerCP-conjugated from BD Bioscience), anti-7AAD (PerCP-conjugated BD Bioscience), anti-CD14 (APC-Cy7 or Percp-conjugated from BD Bioscience), and anti-CD11b (PE-cy7-conjugated from BD Bioscience). The following fluorescence-conjugated antibodies were also used:CD15 (Percp), CD33 (FITC and PE), HLA-DR (APC and PE), CD39 (APC), CD73 (FITC), CD124 (PE), CD56 (PE), CD19 (FITC), Ki-67 (PE), AnnexinV (APC), GranzymeB (PE), IFN-γ (APC), and Perforin (FITC) obtained from BD Biosciences (Supplementary TableS1 ). Among them, GranzymeB, IFN-γ and Perforin were stained intracellularly as follows: cells were first fixed with 2% parafolmaldehyde and permeabilized with 0.1% saponine–PBS buffer. Next, cells were incubated for 15min on ice with antibodies labeled with fluorochrome in the darkness. For surface assessment, cells were incubated with fluorochrome-labeled antibodies directly. In each case, isotypic control was performed. PBMCs were stained according to the manufacturer’s instructions. The appropriate isotype-matched control antibodies were purchased from BD Bioscience. Cells were analyzed using a FACS Canto II flow cytometer (BD, USA) and Diva analysis software (BD).
Diva analysis software
Manufactured by BD
Sourced in United States
Diva analysis software is a data analysis tool designed for use with BD instruments. It provides functionality for processing and analyzing data generated from BD equipment.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (2 mentions)
Hla dr, by BD (1 mentions)
Foxp3 staining buffer set, by BioLegend (1 mentions)
Canto 2, by BD (1 mentions)
Lsrfortessa system, by BD (1 mentions)
Epcam vioblue, by Miltenyi Biotec (1 mentions)
7 amino actinomycin, by BD (1 mentions)
Cd44v3 apc, by R&D Systems (1 mentions)
Facscanto 2, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Fortessa, by BD (1 mentions)
Cd44 pe, by BD (1 mentions)
7 aad, by BioLegend (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
6 protocols using diva analysis software
1
Flow Cytometry Analysis of Immune Cells
2
Multiparametric Flow Cytometry Analysis of Regulatory T Cells
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Fresh human peripheral blood mononuclear cells (PBMCs) were stained with anti-CD4 (PerCP-Cy 5.5 or APC-Cy7-conjugated from BD Bioscience), anti-CD25 (APC-Cy7 or APC-conjugated from BD Bioscience), and anti-CD45RA (FITC-conjugated from BD Bioscience). Intracellular detection of Foxp3 with anti-Foxp3 (PE-conjugated from BD Bioscience) was performed on fixed and permeabilized cells with the Foxp3 staining buffer set (Biolegend, USA) according to the manufacturer's instructions. The following fluorescence-conjugated antibodies were also used: CD39 (APC), Interferon-γ (IFN-γ) (PE-Cy7 or APC), and TGF-β (APC) obtained from BD Biosciences. PBMCs were stained according to the manufacturer's recommendations. The appropriate isotype-matched control antibodies were purchased from BD Bioscience. Cells were analyzed using a FACSCantoII flow cytometer (BD, USA) and Diva analysis software (BD, USA).
3
Phenotyping of Human Peripheral Blood Mononuclear Cells
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Fresh human mononuclear cells were isolated from the peripheral blood using density gradient centrifugation. To identify the cell surface phenotype, the cells were incubated with a primary antibody conjugated to a fluorescent dye. Viable cells (1 × 105) were stained with anti-human CD3, CD8, CD4, CD28, Tim3, and PD-1 antibodies. Dead cells were stained using 7-AAD. Isotype controls were performed on each cell type. The cells were analyzed using flow cytometry (CantoII, BD Biosciences, San Jose, CA, USA) and Diva analysis software (BD Biosciences). During the analysis, the percentage of positive cells was calculated.
4
Differentiation Stage Identification
5
Flow Cytometric Analysis of Tumor Cell Subpopulations
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100,000 cells were stained with Aldefluor® reagent and then with anti-human CD44-PE as previously reported [4 (link), 23 (link)] and CD44v3-APC. Flow cytometry was performed using BD FACSCanto II or Fortessa instruments and DIVA analysis software (BD). FACS-sorted cells were performed after dissociation of PDX tumours with human tumour dissociation enzyme and GentleMACS dissociator (all from Miltenyi). 7-amino actinomycin (BD) positive dead cells were excluded and EpCAM+panCD44+/CD44v3+ and EpCAM+panCD44−/CD44v3− were sorted using FACS Aria II instrument. Antibodies used were 1:100 EpCAM-Vioblue (Miltenyi), 1:20 CD44-APC or CD44-PE (BD clone G44-26) or CD44v9-PE (Biolegend), 1:15 CD44v3-APC (R&D clone 3G5) and 1:50 CD44v6-PE vio770 (Miltenyi).
6
Flow Cytometric Characterization of CD271+ and CD90+ Cells
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The cells were collected and dissociated into single cells suspended in PBS containing 3% FBS. The cells were stained with fluorescence-conjugated monoclonal antibodies against CD271 and CD90 (BD Biosciences) for 20 min in the dark at 4 °C. The corresponding isotype immunoglobulins were used as controls. Dead cells were identified using 7-AAD (Biolegend, San Diego, CA, USA). Data were acquired on a BD FACS Canto II cytometer (BD Biosciences) and analyzed with Diva analysis software (BD Biosciences). Three independent experiments were performed for each analysis.
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