PBMCs were stained with fluorochrome-conjugated antibodies at a density of 2×104 cells per 1 uL of 1× PBS for 30 min on ice. The following fluorochrome-conjugated antibodies were used for flow cytometry antibody staining: FITC anti-human CD8a (clone: RPA-T8), APC anti-human CD69 (clone: FN50), APC/Cy7 anti-human CD4 (clone: RPA-T4), Pacific Blue anti-human CD3 (clone: UCHT1) and PE anti-human HLA-DRB1 (clone: NFLD. D2). All fluorochrome-conjugated antibodies were purchased from BioLegend. PBMCs were fixed with 500 uL of Biolegend Fixation Buffer per 106 cells for 20 min at ambient temperature. The iCyt Synergy SY3200 Cell Sorter (Sony Biotechnology Inc.) was used in conjunction with WinList 9.0.1 software (Verity Software House) for flow cytometry sample processing and analysis, respectively. PBMCs were gated to select for CD3+ CD8+ HLA-DBR1+ T cells (online supplementary figure 1 ). The median fluorescence intensity (MFI) of PE anti-human HLA-DRB1 was measured for the CD3+ CD8+ T cell population and normalised to the background PE anti-human HLA-DRB1 MFI of the unstained PBMC population. The normalised MFI (nMFI) was determined by the following equation: .26 (link) FlowJo version 10 was used to generate MFI histograms (FlowJo LLC).
Winlist 9
Manufactured by Verity Software House
Sourced in United States
WinList 9.0.1 is a software package designed for the analysis and visualization of flow cytometry data. It provides tools for gating, compensation, and data representation.
Automatically generated - may contain errors
Lab products found in correlation
Icyt synergy sy3200 cell sorter, by Sony (1 mentions)
Version 10, by FlowJo (1 mentions)
Live dead fixable violet, by Thermo Fisher Scientific (1 mentions)
Facsdiva acquisition software, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Fitc conjugated annexin 5, by BD (1 mentions)
2 protocols using winlist 9
1
Flow Cytometric Analysis of PBMCs for HLA-DRB1 Expression
2
Annexin V Apoptosis Assay for OVV and Doxorubicin
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Adherent cultures from established cell lines were either infected with OVV at an MOI of 1 for 2 h, incubated with 1 μM DOX (Sigma, St. Louis, MO, USA) for 16 h, or treated with both consecutively. Cultures were detached with Accutase (Sigma, St. Louis, MO, USA) and stained with Live/Dead fixable violet (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s instruction. Cells were subsequently incubated with FITC-conjugated annexin V (BD Pharmingen, San Diego, CA, USA) according to the recommended procedure. Analysis was conducted on an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) with FACSDiva acquisition software (BD Biosciences, San Jose, CA, USA). Data analysis was performed using WinList 9.0.1 (Verity Software House, Topsham, ME, USA).
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