70 μm nylon cell strainer

For obtaining bone marrow, male or female mice were euthanized, and femurs, tibias, and fibulas were excised. Bones were sterilized with 70% ethanol and washed with BMDM media (20% FBS (HyClone), 0.1% β-mercaptoethanol, 1% sodium pyruvate, 10% conditioned supernatant from 3T3 fibroblasts, in DMEM (Gibco) with 4.5 g l^–1 glucose and 100 μg/ml streptomycin and 100 U/ml penicillin), and ground with a mortar and pestle. Bone homogenate was passed through a 70 μm nylon cell strainer (Thermo Fisher Scientific, 08-771-2) for particulate removal. Filtrates were then centrifuged at 290 g in an Eppendorf 5810 R centrifuge for 8 min, supernatant was aspirated, and the pellet was resuspended in BMDM media. Cells were plated in 30 ml BMDM media in non-TC-treated 15 cm petri dishes at a ratio of 10 dishes per two femurs/tibias and incubated at 37°C. An additional 30 ml of BMDM media was added 3 d later. At 7 d the media was aspirated, 15 ml cold PBS (Gibco, 10010–023) was added, and cells were incubated at 4°C with for 10 min. BMDMs were scraped from the plate, collected in a 50 ml conical tube, and centrifuged at 290 g for 5 min. PBS was aspirated, and cells were resuspended in BMDM media with 30% FBS and 10% DMSO at 10⁷ cells/ml. One milliliter of aliquots was stored at –80°C for 24 hr in Styrofoam boxes and then moved to long-term storage in liquid nitrogen.

We isolated AG⁺ hPGCLCs from floating aggregates by using FACS. Floating aggregates containing hPGCLCs, which were induced from hiPSCs, were dissociated into single cells through 0.1% trypsin/EDTA treatment for 15 min at 37°C with periodic pipetting. After the reaction was quenched by addition of an equal volume of fetal bovine serum, cells were resuspended in FACS buffer (0.1% BSA in PBS) and strained through a 70 μm nylon cell strainer (Thermo Fisher Scientific) to remove cell clumps. AG⁺ cells were sorted with a FACSAria Fusion flow cytometer (BD Biosciences) and collected in an microcentrifuge tube containing CELLOTION (Amsbio). All FACS data were collected in FACSDiva v8.0.2 software (BD Biosciences) and analyzed with FlowJo v10.8.1 (BD Biosciences). For flow cytometry analysis, hPGCLCs were dissociated and resuspended in FACS buffer. AG⁺ cells were analyzed with FACSCanto or FACSFortessa (BD Biosciences).