2.3. Isolation and culture of mouse embryonic fibroblasts

Isolation and culture of mouse embryonic fibrobalsts (MEFs) were performed according to the protocol of Jozefczuk et al. that previously was described (Hormozi, Assaei, & Boroujeni, 2017; Jozefczuk, Drews, & Adjaye, 2012). In brief, pregnant mouse was sacrificed 13 d.p.c (day postcoitum) by cervical dislocation. Uterine horns were dissected, rinsed with 70% (v/v) ethanol and soaked in PBS buffer w/o Ca/Mg ions. The experimental procedures were carried out under sterile conditions in a tissue culture hood. The uterine horns were placed in Petri dish and each embryo was separated from its embryonic sac and placenta. The tissue is chopped with carpels into smaller fragments, 1 ml of 0.05% trypsin/EDTA (Gibco, Invitrogen) containing 100 Kunits of DNase I was added to each embryo. The tissue was transferred into a 50 ml falcon tube and incubated for 15 min at the room temperature. Cells were dissociated every 5 min by pipetting thoroughly. Freshly prepared MEF medium was used for inactivating trypsin. The lysate was centrifuged at low speed (300× g) for 5 min, the cell pellet was discarded and supernatant was suspended in a fresh MEF medium. A number of cells equivalent to 3–4 embryos in each T150 (TPP) flask coated with 0.2% of bovine gelatin (Gelatin from bovine skin, type B, Sigma) were plated for 2 hr. At this time, the fibroblasts (passage 0) were the only cells capable of attachment to the gelatin‐coated flasks. The cells were allowed to reach 80%–90% confluency at this level; the P0 cells were trypsinized and stored at −70C for future usage. Other cells in the flask were allowed to reach P3 or P4 before use.