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EMSC Isolation and Culture

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