Cd14 pe cy5

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).

Absolute counts of peripheral blood leukocytes were determined for all study participants using BD Trucount tubes, six-color TBNK Reagent and, in addition, CD123 BUV395 (BD Biosciences), CD15 PB, CD193 BV605, and HLA-DR BV785 (Biolegend), and CD14 PE-Cy5 (eBioscience) according to the manufacturer’s instructions. Samples were fixed in 1× BD fluorescence-activated cell sorter (FACS) lysing solution (BD Biosciences) for 2 h and acquired on a BD FACSymphony A5 instrument, equipped with ultraviolet (355nm), violet (405 nm), blue (488 nm), yellow/green (561 nm), and red (637 nm) lasers. For absolute cell count calculations, the number of events for populations of interest was divided by the number of bead events and multiplied by the BD Trucount bead count.