Peripheral blood mononuclear cells from 15 patients [mean age 48.5 (range 22–78) years, 53% females] and 15 healthy controls [mean age 46.8 (22–68) years, 53% females] were thawed, stained with Live/Dead Fixable Yellow Dead Cell Stain Kit according to the manufacturer’s instructions (Invitrogen, cat. L34959) and Fc-blocked with BD Pharmingen Human BD Fc Block (BD, cat.564219). Subsequently, cells were stained with the following antibodies: V500 anti-human CD3, PerCP-Cy5.5 anti-human CD4, PE-Cy5 anti-human CD8, PE-Cy7 anti-human CD25, APC-H7 anti-human CD45RA, BV421 anti-human CTLA4, BV785 anti-human CD31, BV650 anti-human HLA-DR, biotin anti-human TCR γ/δ, biotin anti-human CD1c, and biotin anti-human CD14. Cells were then stained with FITC Streptavidin (BioLegend, 1:500, cat. 405202; Supplementary Table 1 ). Fixation and permeabilization were achieved using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, cat. 00-5523-00) according to instructions from the manufacturer. Cells were intracellularly stained with APC anti-human Helios and PE-CF594 anti-human FOXP3 (Supplementary Table 1 ). Cells were analysed using the BD LSRFortessa Cell Analyzer and the BD FACSDiva Software. FlowJo v10.2 (BD) was used to analyse flow cytometric data.
Pharmingen human fc block
Manufactured by BD
The BD Pharmingen Human BD Fc Block is a laboratory reagent designed to block Fc receptors on cells. It is used to prevent non-specific binding of antibodies during flow cytometry and other immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Streptavidin fitc, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Live dead fixable yellow dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Cytoflex lx device, by Beckman Coulter (1 mentions)
2 protocols using pharmingen human fc block
1
Comprehensive Phenotyping of Immune Cells
2
Quantifying Cell Surface Antigens by Flow Cytometry
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Cells were counted and their viability was assessed using trypan blue staining. They were then seeded in a 96 V-well plate at a density of 100,000 cells/well. They were first incubated with BD Pharmingen™ Human BD Fc Block™ (#564219, BD Biosciences) for 10 min at room temperature. They were then washed using PBS 2% BSA and incubated with primary antibodies for 60 min at 4 °C. They were then washed once again and incubated with secondary antibodies and/or primary fluorochrome-coupled antibodies for 60 min at 4 °C. They were washed again before being fixed using 2% paraformaldehyde (ThermoFisher). They underwent a final wash and were resuspended in 200 μL of PBS 2% BSA. Fluorescence was measured using the CytoFLEX LX device (Beckman Coulter) and data analyzed using the Kaluza software (Beckman Coulter). Antibodies used are listed in Supplementary Table 3 . Raw flow cytometry data is available at the end of the Supplementary Data Appendix.
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