Crl cd1

Female BALB/c mice (BALB/cAnNCrl, (7 ± 1 weeks old, body weight 18 ± 1 g)), female CD-1 mice (Crl:CD-1, (7 ± 1 weeks old, body weight 27 ± 1 g)), and female NMRI mice (Crl:NMRI, (7 ± 1 weeks old, body weight 20 ± 1 g)) were obtained from Charles River (Sulzfeld, Germany, or Raleigh, NC, USA). Female and male C57/BL/6 mice (C57/BL/6J, (7 ± 1 weeks old, body weight 20 ± 1 g)), female and male TRPV1^−/− mice (B6.129X1-Trpv1^tm1Jul/J, (7 ± 1 weeks old, body weight 20 ± 1 g)) and female and male TRPA1^−/− mice (B6;129P-Trpa1^tm1Kykw/J, (7 ± 1 weeks old, body weight 20 ± 1 g)) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All animals were kept in groups of four or six mice per cage with a 12 h light/dark cycle at 22 °C. Water and a standard diet (Altromin 1824/LabDiet 5001) were provided ad libitum. The animal experiments have been ethically approved by the LAVES institute, Oldenburg, Germany (AZ 33.12-42502-04-16/2213, approval date: 28.09.16) and by the North Carolina State University Animal Care and Use Committee (IACUC Protocol No. 16-154-B and 16-038-B (1)).

RBCs and CBC2s were counted in C57BL/6J (hereafter B6/J) and A/J mice, and in 26 genetically distinct recombinant inbred (RI) strains derived from them comprising the AXB/BXA strain set. The cell count data from these mice have previously been reported in an on-line appendix (Keeley et al., 2014b (link)), while a more extensive study has recently reported the cell counts and quantitative trait locus (QTL) analysis for the RBC population (Kautzman et al., 2018 (link)); the methodology associated with these analyses is briefly recapitulated below. Eyes from Xkr8 knockout mice (KO) and heterozygous (Het) littermate control mice (wild type littermate controls were not available) were provided by the laboratory of Dr. Shigekazu Nagata at Osaka University in Japan (Suzuki et al., 2013 (link)). Electroporation experiments were conducted on CD1 mice originally obtained from Charles River Laboratories (Crl:CD1, #022), and subsequently bred in the UCSB Animal Resource Center. B6/J and A/J mice were also bred in-house, and used for in situ hybridization, immunofluorescence and qPCR studies. All experiments were carried out under authorization by the Institutional Animal Care and Use Committee at UCSB, and in accord with the NIH Guide for the Care and Use of Laboratory Animals.

To generate Ildr2 floxed mice (Ildr2^fl/fl), a targeting construct containing Ildr2 exon 1 flanked by loxP sites and a neomycin resistance cassette (transcribed in opposite direction of gene, 1.6 kb from exon 1) was electroporated into V6.5 (129 x B6 F1) ESCs. Electroporated ESCs were selected with G418, picked, and 384 colonies were clonally expanded. Positive integration of the targeting construct was identified by PCR followed by confirmation of correct integration by Southern blot. A positive clone was injected into E3.5 BDF1 blastocytes and transferred into the uteri of pseudopregnant CD1 mice (Crl:CD1; Charles River). Floxed alleles in the pups were confirmed by PCR. Chimeric mice were crossed with C57BL/6J (Jackson Labs) mice and offspring were confirmed carriers by PCR and Southern blot analysis. Mice that carried the floxed allele were backcrossed to C57BL/6J mice (obtained from Jackson Labs) for 10 generations then intercrossed to create B6.129S-Ildr2^fl/fl mice. These mice were bred with albumin-Cre mice (B6.Cg-Tg(Alb-cre)21Mgn/J, Jackson Labs stock #003574) until all offspring segregated for two floxed alleles and one or no copies of Cre.