Isolation and Quantification of Murine Basophils

Single-cell suspensions of bone marrow (BM) cells (in PBS containing 2% FCS and 2 mM of EDTA) were stained with biotin-conjugated anti-Ly6G (1A8) and anti-CD19 (6D5) antibodies (BioLegend, San Diego, CA, USA) before removing positive cells with MagniSort™ streptavidin-negative selection beads following the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). After this basophil enrichment, primary BM basophils were selected for CD200R3 expression and sorted using a BD FACSMelody™ cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). RNA extraction was performed by using Trizol reagent as described in the manufacturer’s protocol (Invitrogen). cDNA was synthesized with SuperScript™ III First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed with SsoAdvanced SYBR green reaction mix (Bio-Rad, Hercules, CA, USA) using the M_B2m_1 KiCqStart™ primer pair for mouse β2-microglobulin as housekeeping gene (Sigma-Aldrich, Merck) and the following primers for Mcpt8 quantification: Forward primer: 5′-GTGGGAAATCCCAGTGAGAA-3′ and Reverse primer: 5′-TCCGAATCCAAGGCATAAAG-3′ (12 (link)). Quantitative PCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and, following amplification, Ct values were obtained using the CFX Manager™ software 2.1 (Bio-Rad, Hercules, CA, USA).