Anti mouse cd19 antibody

Splenocytes were sorted by flow cytometry using anti-mouse CD19 antibody (BioLegend, San Diego, CA). Cells were washed with 1x PBS and mRNA was extracted using an RNeasy RNA extraction kit (Qiagen, Valencia, CA) following the manufactures protocol. Messenger RNA was reverse-transcribed to cDNA using an iScript Supermix synthesis kit (Bio-Rad, Hercules, CA) following the manufactures recommendations. Gene expression was quantified by RT-qPCR as described previously [21 (link)]. The primers for detecting expression of Nek2 are TTC CAT CCT CAG CCA TGA AGA and CCT GCA CTT GGA CTT GGC AA.

C57BL/6J mice were treated with CpG ODN (10 μg/mouse) or each LNP-CpG (10 μg CpG ODN/mouse) subcutaneously at the base of the tail. Twenty-four hours after administration, the lymph nodes draining the site of administration were collected after euthanasia. To prepare single-cell suspensions, the draining lymph nodes were incubated with 200 μg/mL Liberase TL (Roche Diagnostics GmbH, Mannheim, Germany) and 10 U/mL DNase I (Roche Diagnostics GmbH) for 60 min at 37°C. Prepared cells were incubated with anti-mouse CD3ε antibody (BioLegend), anti-mouse CD19 antibody (BioLegend), anti-mouse CD16/CD32 antibody, anti-CD11c antibody, anti-PDCA-1 antibody (BioLegend), anti-CD80 antibody, or anti-CD86 antibody for flow cytometry. In this way, the DCs were separated into two subsets, namely CD3ε⁻ CD19⁻ PDCA-1⁺ CD11c⁺ pDCs and CD3ε⁻ CD19⁻ PDCA-1⁻ CD11c⁺ conventional DCs (cDCs). The cells were then analyzed by means of flow cytometry.