Weigh tissue and transfer to 100mm plate with cold HBSS with 1% BSA.
Use autoclaved scissors and forceps to cut tissue into small enough pieces to be passed through a 10ml pipette.
Transfer tissue pieces and HBBS to a conical tube.
Spin at 1800rpm for 5 min at 4°C.
Aspirate supernatant and resuspend in 30mls cold PBS. Repeat wash.
Add enzymatic digestion solution at 10ml/g of tissue (Note: fill to less than 90% capacity) 0.8–1.5mg/mL Collagenase I (depending on the batch), 10μM ROCKi (optional), 0.2mg/ml DNaseI, 10μl/mL Fungizone, 10μl/mL Pen/Strep in HBSS
Digest overnight at 37°C with rocking on an Adams Nutator mixer.
Spin at 1800rpm for 5 min at 4°C. (Note: floating debris is normal and discarded)
Wash 2X in 25ml cold PBS.
Resuspend cells in 5 ml TrypLE and incubate for 5 min on Nutator mixer at 37°C.
Add to TrypLE 15 mls DMEM + 10% FBS with 1mg/ml DNaseI.
Spin at 1800rpm for 5 min at 4°C.
Resuspend in 10ml DMEM + 10% FBS and pass through 18-G needle/10ml syringe 2–5X. Repeat with 20-G and 22-G needles. (Note: if tissue cannot be passed through 18-G needle, first proceed to step 14, forcing through needles decreases viability)
Filter cells/media through 100μm cell strainer and wash conical tube with additional 10 ml DMEM + 10% FCS.
Spin at 1800rpm for 5 min at 4°C.
Resuspend in 5mls of 1X RBC lysis buffer (10x plus distilled water) for 5 min on ice with occasional shaking.
Add 25 ml cold PBS and spin at 1800rpm for 5 min at 4°C.
Resuspend pellet in 10 ml DMEM + 10% FBS and filter through 40μm cell strainer. Wash with addition 10 ml DMEM + 10% FBS.
Spin at 1800rpm for 5 min at 4°C.
Resuspend cell pellet with 10ml DMEM + 10% FBS for counting with trypan blue.
Spin down and resuspend 10million cells/ml in 10% DMSO, 90% FBS for freezing in liquid nitrogen.
