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Pd 1 bv605 clone eh12.2h7

Manufactured by BioLegend

PD-1 BV605 (clone EH12.2H7) is a fluorochrome-conjugated antibody product manufactured by BioLegend. It is designed for the detection and analysis of PD-1 expression on cell surfaces.

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2 protocols using pd 1 bv605 clone eh12.2h7

1

HIV-Flow Assay for Enriched CD4+ T Cells

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Enriched CD4+ T cells were processed for HIV-Flow assay50 (link). Briefly, after 1 h pre-incubation with 5 μg/ml Brefeldin A (BFA) and 24 h stimulation with of 162 nM PMA and 1 µg/ml ionomycin in the presence of ARVs (200 nM lamivudine and 200 nM raltegravir), extracellular staining was performed using the following antibodies: Live/Dead Aqua Cell Stain (ThermoFisher Scientific cat.L34957), CD45RA APC-H7 (clone HI100; BD cat.560674), CCR7 BB700 (clone 3D12; BD cat.566437), PD-1 BV605 (clone EH12.2H7; Biolegend cat.329924), TIGIT eF450 (clone MBSA43; eBioscience cat.48-9500-42), HLA-DR AlexaFluor700 (clone G46-6; BD cat.560743), ICOS BV785 (clone C398.4 A; Biolegend cat.313534), α4/CD49d PE-Cy7 (clone 9F10; Biolegend cat.304313) and β1/CD29 BB515 (clone MAR4; BD cat.564565). Cells were simultaneously fixed and permeabilized with the FoxP3 Buffer Set (eBioscience), followed by intracellular staining of HIV p24 with clone 28B7 APC (MediMabs cat.MM-0289-APC) and clone KC57 PE (Beckman Coulter cat.6604667). All samples were resuspended at a final concentration of 1 × 106 cells/ml in PBS and filtered prior to cell sorting.
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2

Flow Cytometry and Single-Cell Sorting of Activated Circulating T Follicular Helper Cells

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For flow cytometric cell analyses and cell sorting, freshly thawed and washed PBMCs (5x10 6 ) were incubated for 30-60 min at 4 °C in the dark with 100 µl staining cocktail, containing the following fluorescently-labelled antibodies diluted in FACS buffer (2% FBS in PBS): CD4-APC-Cy7 (clone A161A1; BioLegend), CD8a-Alexa700 (clone SK1; BioLegend); CD45RA-BV510 (clone HI100; BioLegend); PD-1-BV605 (clone EH12.2H7; BioLegend), ICOS-PE-Cy7 (clone C398.4A; BioLegend), CXCR5-Alexa647 (clone RF8B2; BD Biosciences), CXCR3-BV421 (clone G025H7; BioLegend), CCR6-PE (clone G034E3; BioLegend) and CCR7-BV710 (clone G043H7; BioLegend). Subsequently, cells were washed with FACS buffer and incubated with the live-dead marker 7-aminoactinomycin D (7-AAD; Thermo Fisher Scientific) for 10 min 4 °C in the dark. Flow cytometric analyses and indexed single-cell sorting were performed using a FACS AriaIII (BD Biosciences) and FACSDiva software (version 8.0.1) as described 19 . In brief, single activated circulating 7-AAD -CD3 + CD4 + CD45RA -CXCR5 + PD-1 hi ICOS + cTFH were sorted into 384-well plates containing 2 μl random hexamer primer (RHP) mix (consisting of nuclease free water, PBS, RNAsin, NP-40, DTT, random hexamer primers), immediately sealed, frozen on dry ice, and stored at -80 °C until further processing.
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