Cd19 apc cy7

Spleens were prepared and stained as described [21] (link) using the following surface antibodies for cell enumeration: biotinylated Siglec H (Hycult Biotech) and streptavidin-APC (BD Biosciences), CD11c PE-Cy7 (BD Biosciences), CD11b PerCP-Cy5.5 (Biolegend), CD19 APC-Cy7 (BD Biosciences), CD3e Pacific Blue (Biolegend), Ter-119 FITC (Biolegend). For intracellular cytokine staining the following antibodies were used: biotinylated Siglec H (streptavidin-APC), CD11c PE-Cy7, CD11b PerCP-Cy5.5, CD19 APC-Cy7, TNF Alexa 700 (BD Biosciences), IL-12p40 PE (BD Biosciences), IFNalpha FITC (Antigenix).

Cryopreserved 10⁷ PBMC were thawed into 1 ml preheated RPMI1640, centrifuged at 300 × g for 5 min, resuspended in 500 μl FACS buffer (PBS + 2% FBS), and incubated with 200 nM his-tagged antigen (gH/gL) for 45 min at 4 °C. The PBMC were then washed two times with 1 ml FACS buffer and resuspended in 100 μl FACS buffer. The PBMC were stained with the following antibodies: CD3-PE-Cy5 (BD Biosciences Cat#555341) at a 1:25 dilution, CD14-PE-Cy5 (eBioscience Cat#15-0149-42) at a 1:50 dilution, CD16-PE-Cy5 (BD Biosciences Cat#555408) at a 1:25 dilution, CD235a-PE-Cy5 (BD Biosciences Cat#559944) at a 1:100 dilution, CD19-APC-Cy7 (BD Biosciences Cat#348794) at a 1:100 dilution, CD20-PE-Cy7 (BD Biosciences Cat#335793) at a 1:200 dilution, IgG-FITC (BD Biosciences Cat#555786) at a 1:25 dilution, and anti-his-PE (BioLegend Cat#362603) at a 1:20 dilution for 30 min at 4 °C. The PBMC were washed three times with 1 ml FACS buffer and resuspended in 500 μl FACS buffer, then subjected to FACS on a BD FACS Aria II (BD Biosciences).
Antigen-positive B cells (CD3-, CD14-, CD16-, CD235a-, CD19+, CD20+, IgG+, PE+) were sorted individually into 96-well PCR vital-plates containing 20 μl first strand buffer (5 μl first strand buffer, 0.5 μl of RNase inhibitor (Invitrogen Cat#10777019), 1.25 μl of 100 μM DTT, 0.06 μl of IGEPAL (Sigma Cat#I8896).