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OBJECTIVE_ Split HeLa Cells


									OBJECTIVE: Split HeLa Cells

500ml DMEM @4C
50ml Fetal Bovine Serum (FBS), frozen
5ml Pen-Strep. frozen
5ml L-Glu, frozen
Trypsin, frozen
Plate of cells to be split
Empty plates, as necessary

Thaw above ingredients in water bath

Media Preparation:
  1. Spray down bio hood area and unopened bottles with 70% EtOH for sterilization
  2. Combine FBS, Pen-Strep, and L-Glu in DMEM bottle
  3. Stir thoroughly, do not invert
  4. Label DMEM bottle: DMEM, FBS, P/S, L-Glu, [Name], [Date]
  5. Dispose of pipettes, used bottles in biohazard

Cell Preparation:
   1. Use aspirator with a Pasteur pipette to remove media from dish
   2. Quickly add 4ml Trypsin
   3. Mix by pipetting up and down; break up cell clumps if possible
   4. Dilute mixture in 6ml media
   5. Centrifuge at 1000rpm for 5 minutes
   6. Drain off liquid
   7. Resuspend pellet in 10ml media

Final preparations:
   1. Label empty plates – HeLa, Passage #, initials, dilution, date prepared
   2. Add media and cell mixture according to dilution:
        Dilution                   Resuspension volume          Media volume
        5:1                        2ml                          8ml
        10:1                       1ml                          9ml
        20:1                       0.5ml                        9.5ml
   3. Swirl, rock cells around plate
   4. Store in 37C Incubator
   5. Serve lukewarm, best with a dry Cabernet or Merlot

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