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Cd19 apccy7

Manufactured by Miltenyi Biotec
Sourced in Germany

The CD19 APC-Cy7 is a fluorochrome-conjugated antibody that binds to the CD19 antigen. CD19 is a cell surface antigen expressed on B cells and B cell precursors. The APC-Cy7 fluorochrome provides a bright and stable signal for flow cytometric detection and analysis.

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2 protocols using cd19 apccy7

1

PBMC Isolation and Multicolor Flow Cytometry

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PBMCs isolated from HDLS donor blood were seeded in round bottomed 96‐well plates in Roswell Park Memorial Institute (RPMI) media with 10% FBS at 4 × 105 cells per well and incubated in a 5% CO2 incubator at 37°C overnight. Cells were centrifuged at 300 g for 5 min and blocked for 10 min with 50 μl 10% human AB serum, 1% FBS in PBS. Blocked PBMCs were incubated in Live/Dead Aqua (1:500; Life technologies (Fisher/Invitrogen) L34957 ) for 30 min, washed in PBS and incubated in fluorochrome‐labelled primary antibody diluted 1:10 in PBS, 1% FBS for 20 min at 4°C. (Lin FITC (BioLegend 348801), CD3 PerCP (Miltenyi 130‐100‐458), CD19 APCCy7 (Miltenyi 130‐098‐073), CD14 PacBlue (Miltenyi 130‐098‐058). Cells were washed twice with PBS 1% FBS and analysed by flow cytometry immediately. Gating during analysis was based on fluorescence minus one controls. Flow cytometry was carried out on a BD LSRFortessa cell analyser and analysed using FlowJo software (Tree Star).
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2

Calcium Signaling in NKG2A-Educated NK Cells

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NK cells were enriched from freshly isolated PBMCs using an EasySep Human NK Cell Enrichment Kit (Stemcell, Vancouver, Canada) as per the manufacturer's guidelines and sorted using a BD FACS Aria-Fusion. NK cells were labeled with CD3-AF700, CD14-APC-Cy7, CD19-APC-Cy7, NKG2A-APC (Miltenyi Biotec, Bergisch Gladbach, Germany–REA110), LIVE/DEAD fixable near-IR dye and a combination of KIR2DL1/KIR2DS5-PE, KIR2DL2/S2/L3-PE (BioLegend–DX27), and KIR3DL1-PE (BioLegend–DX9) depending on specific donor genotype for 20 min at 4°C in the dark. Cells were sorted to select for NKG2A-educated NK cells and uneducated NK cells while excluding KIR-educated NK cells and were subsequently incubated overnight in RPMI containing 10% (v/v) FBS with 1 ng/mL IL-15. NK cells were washed then resuspended in calcium buffer (HBSS + 1 mM CaCl2 + 0.5 mM MgCl2 + 10 mM HEPES + 0.1% FBS) and stained with 4 μM Fura2 (Life Technologies, CA, USA) for 30 min at 37°C. Cells were resuspended in calcium buffer then observed under a fluorescent microscope. Biotinylated anti-NKp46 (BioLegend–9E2) and anti-2B4 (BioLegend–C1.7) antibodies were added after 3 min then streptavidin (BioLegend) after a further 2 min. Thapsigargin (Merck Millipore, MA, USA) was added at 17 min. The 340/380 ratio of Fura2 was recorded to measure intracellular calcium release by cells over time at 37°C using an incubator chamber.
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