Anti cd19 bv605

Quadriceps muscles from immunized mice were harvested after 18–24 h of the injection and cut into small pieces. A single cell suspension was prepared using skeletal muscle dissociation kit (Miltenyi Biotec) according to manufacturer’s protocol. Briefly, small pieces of muscle were incubated in enzyme solution (enzyme D, enzyme P and enzyme A in DMEM) for 30 min at 37^oC with gentle shaking, passed through 70 µm cell strainer and wash with 10 ml DMEM. Centrifuge cell suspension at 300xg for 20 min. Cells were stained with a combination of the following fluorescently labeled antibodies: anti-CD45-AF700, anti-CD3-BV786, anti-CD19-BV605, anti-CD49b-PE, anti-CD11b-PE-Cy5, anti-CD11c-BV711, anti-F4/80-FITC, anti-Ly6C-PerCp, anti-Ly6G-PE/Cy7, anti-MHCII-PE/Dazzle594, anti-CD80-BV650, anti-CD40-APC, anti-CXCR3-BV510 and anti-CCR2-BV421 (all from BioLegend). Live/dead cells were analyzed using fixable viability dye efluor780 (eBioscience). The stained cells were acquired using BD LSRFortessa cell analyzer and data was analyzed using FlowJo software (Tree Star). Total immune cells (CD45⁺), myeloid cells (CD11b⁺), monocytes (CD11b⁺Ly6C⁺), macrophages (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Ly6G⁺), dendritic cells (DCs; CD11c⁺MHCII⁺), natural killer cells (NK cells; CD49b⁺, CD3⁻), B cells (CD11b⁻CD11c⁻CD19⁺), T cells (CD11b⁻CD11c⁻CD3⁺) and NKT cells (CD49b⁺CD3⁺) were gated by flow cytometry.

This genotyping was done for all donors and was the sole method used for a few of the control donors. The homozygous F/F genotype was distinguished from F/V & V/V genotypes using the MEM-154 clone of anti-CD16 mAb (PE-labeled, Pierce Chemical Co, Rockford, IL). MEM154 reacts with the CD16A 158 V but not the 158 F [36 (link)). PBMCs were labeled with: FITC-anti-CD3e (cloneOKT3); PE-anti-CD16A (3G8) or PE anti-CD16A 158V selective-(MEM154); BV605-anti-CD19 (HIB19.11); PacBlue anti-CD45 (HI30); FITC-anti-CD91 (2MR-alpha) and APC-Cy7-anti-CD56 (HCD56), purchased from BioLegend (San Diego, CA) with the exception of MEM154. CD16A F/F cells were negative with clone MEM154 and positive with clone 3G8.