Prepare: -HBSS (Invitrogen #14025-092) Add 100 μl Primocin (Invivogen #ant-pm-1) per 50 ml HBSS Filter with 0.22 μm -Collagenase I (Worthington Biomedical Corporation #LS004196) 2mg/ml Collagenase in sterile HBSS Filter with 0.22 μm -Red Blood Cell Lysing Buffer (Sigma #R7757) -Autoclave all needed tools Isolate Cells: 1) Sacrifice mice and submerge collected ears only into 6 well plates with 70% ethanol 2) Collect external ears and put in sterile HBSS + antibiotics Perform remaining steps under the hood: 3) Was ears with sterile HBSS + antibiotics in the 6 well plates. 3 ml per well 4) Using sterile scissors and razor blades, cut ears into tiny pieces in collagenase solution. It will be easier to cut if you use a small amount of collagenase to cut the ears and then add the minced ears to the remaining collagenase. 5) Digest for 1 hour at 37 ̊C in a water bath. Shake every 15 minutes. 6) Filter through 70 μm cell strainer to remove extra unwanted pieces. (BD Biosciences #352350) 7) Centrifuge at 1350 rpm for 8-9 minutes 8) Discard supernatant 9) Resuspend the pellet in red blood cell lysing buffer following Sigma’s instructions. Be gentle! Add 1 ml of buffer, pipette to mix, let sit for 1 minute, add 10 ml cell culture medium and pipette it again to mix. 10) Centrifuge at 1350 rpm for 8-9 minutes 11) Discard supernatant 12) Resuspend the pellet in culture medium (EMSC + Primocin) and seed (Seed cells from 6 animals/12 ears in one 100 mm dish) 13) Change medium every 2-3 days. Split Cells: 1) Remove medium from wells 2) Wash subconfluent primary cultures with PBS (Invitrogen #10010-023) 3) Remove PBS 4) Add 0.25% Trypsin (Invitrogen #25300-054) 2-3 ml to a 100mm dish 5) Place in incubator for about 3 minutes until cells detach 6) Add culture medium to neutralize the trypsin 6-8 ml to a 100mm dish 7) Collect and centrifuge at 1350 rpm for 8-9 minutes 8) Discard supernatant 9) Resuspend the pellet in culture medium and seed Cells are typically diluted 1:5 or 1:6 at each passage Culture Medium: DMEM/F12 (Invitrogen #11330-032) + 15% FBS (Invitrogen #10082-147) + 100 μL Primocin (Invivogen #ant-pm-1) per 50 mL Media |
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