MEF (C57BL/6) [MEF-BL/6-1]

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. 
2. Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial’s contents plus 5 mL of completes DMEM to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete DMEM to bring the total volume to 10 mL.
4. Gently mix and pellet the cells by centrifugation at 270 x g for 5 minutes.
5. Discard the supernatant and resuspend the cells with 10 mL fresh growth medium (warm) and plate the cells at seed density of 1X10⁴ cells/cm².
6. Add fresh growth medium (warm) to the appropriate size flask.
7. Incubate 37°C in a 5%CO₂ in air atmosphere.
8. Fluid change twice a week or when pH decreases.

 It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 1XPBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
3. Add Trypsin-EDTA (0.25% Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to the flask (Table 1) and incubate for 2 minutes. Gently tapping the flask, observe cells under an inverted microscope. Cells usually detach in 2 to 3 minutes.
4. Add an equal volume complete of the growth medium (Table 1) and rinse surface of the flask to detach all the cells. Gently pipetting up and down will break cell clumps. 
5. Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 x g for 5 minutes.
6. Remove and discard the supernatant
7. Add 10 mL complete growth medium to the cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
8. Add more complete growth medium (Table 1) to the cell suspension as needed to plate cells at approximately 0.8 X 10⁴ cells/cm².
9. Place flasks in the incubator @ 37°C with a 5% CO₂ in air atmosphere 

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 5th edition, published by Alan R. Liss, N.Y., 2005.  

Subcultivation Ratio: 1:5 to 1:8

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