Isolation of Primary Mouse Cell Types

Primary Mouse Embryonic Fibroblasts (MEFs) were generated from E13.5 embryos. After removing the placenta, yolk sac, head and the dark red organs, embryos were finely minced and digested for 20 min in 0.25% trypsin. Single cell suspension was then obtained by pipetting up and down the digested embryos. Mouse Dermal Fibroblasts (MDFs) were isolated as described in (Etemadi et al., 2015 (link)). To generate Bone Marrow Derived Macrophages (BMDMs), bone marrow cells from tibia and femur of 2 month old mice were seeded in non-coated Petri dishes and cultured for 6 days in Dulbecco’s modified Eagle medium + 10% fetal bovine serum + 20% (v/v) L929 mouse fibroblast conditioned medium. Keratinocytes were isolated as described in (Lichti et al., 2008 (link)). Splenocytes were isolated from 2 month old mice. Mouse spleens were mashed through a cell strainer into the Petri dish using the plunger end of a syringe. Cells were then washed once in cold PBS and treated with 1X Red Blood Cell Lysis Buffer (BioLegend, Cat N 420301) for 5 min on ice. Cells were then washed again in PBS and counted.