Ssoadvanced sybr green reaction mix

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).

RNA extraction on re-sorted cells was performed as described in the manufacturer’s protocol (RNeasy Mini kit, Qiagen). cDNA was synthesized with iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed with SsoAdvanced SYBR green reaction mix (Bio-Rad) using the following KiCqStart^TM Primers pairs (purchased from Sigma-Aldrich): M_Actb_1; M_Batf_1; M_Bcl6_1; M_Gata3_1; M_Maf_1, M_Prdm1_1, and M_Cxcr5_1 to respectively measure β-actin, Batf, Bcl6, Gata3, Maf, Prdm1, and Cxcr5 mRNA expression levels. Bach2 primers were purchased from Integrated DNA Technology. All sequences are reported in supplementary Table 3. Quantitative PCR was performed on the CFX96 Touch Real Time PCR Detection System (Bio-Rad) and following amplification, Ct values were obtained using the CFX Manager^TM software 3.1 (Bio-Rad).