Knockout sr

HEK 293 cells (ATCC) were cultured using standard conditions in media containing: DMEM (Life Technologies), 10% FBS (Applied StemCell), and Penicillin-Streptomycin (Life Technologies).
ES cells were cultured using standard conditions in media containing: DMEM (Life Technologies), 7.5% ES-sure FBS (Applied StemCell), 7.5% KnockOut SR (Life Technologies) Penicillin-Streptomycin (Life Technologies), Glutamax (Life Technologies), HEPES buffer (Life Technologies), 2-mercaptoethanol (Life Technologies), and MEM-NEA (Life Technologies). LIF was replaced daily and ES cells were passaged every 48 h.
For long recruitment experiments (>1 h), cells were treated with 3 nM RAP (Selleckchem) and/or 3 nM FK506 (Abcam) added directly to culture dish and changed daily. For short recruitment experiments (≤1 h), cells were treated with 30 nM RAP and/or FK506 in suspension.

Exosomes were extracted from iPSCs using total exosome isolation reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s instructions. Briefly, iPSCs were cultured in 6-well plates, until 80% confluence, in serum-free iPSC growth medium containing Dulbecco’s modified eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham 1:1 (Biological Industries, Beit Haemek, Israel) containing 20% KnockOut SR (Life Technologies, Waltham, MA, USA), 1% nonessential amino acid, 1% L-glutamine (Biological Industries, Beit Haemek, Israel), 0.2% 2-mercaptoethanol (Life Technologies, Waltham, MA, USA), and rhFGF basic (4 ng/mL) (R and D Systems, Minneapolis, MN, USA), at 37 °C, 95% air, and 5% CO₂ in an incubator. Culture medium was then collected and centrifuged at 2000× g for 30 min. The supernatant was then transferred to a new tube and mixed well with 5 mL of total exosome isolation reagent. The homogenous solution was incubated at 4 °C overnight and then centrifuged at 4 °C at 10,000× g for 1 h. The supernatant was then aspirated, and the exosomes were suspended in 500 µL PBS and stored at −80 °C until use. Extracted exosomes were analyzed for particle visualization and rapid, automated analysis of size distribution and concentration by nanoparticle tracking system (NanoSight NS300, Weizmann institution, Rehovot, Israel). Exosomes were saved at −80 °C for following experiments.

Male and female Oct4-GFP(ΔPE) ESCs and female Gcnf^−/− ESCs [8] (link) were grown on gamma-irradiated mouse embryonic fibroblasts (MEFs) in KNOCKOUT DMEM medium containing 4.5 g/l glucose and supplemented with 15% KNOCKOUT SR (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 µM nonessential amino acids (Invitrogen), 1 µM 2-mercaptoethanol (Invitrogen), and 50 µg/ml each penicillin and streptomycin (Invitrogen) in the presence of 1,000 U/ml murine leukemia inhibitory factor (LIF) (ESGRO; Chemicon).