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Propagation of WIF-B cells (2-28-05 update)
General Comment: We have revised our culture conditions to minimize cell
death and the “crashing phenomenon”. Using the protocol below, the cells
plated on coverslips now survive at least 14 days and sometimes longer, maximum
polarity is observed between 12 and 14 days.
- Medium: Complete F-12 Coon’s Modification with 5% Serum and
- Trypsin: Any Commercial product approved for tissue culture
- Coverslips: No 1 1/2, 22mmx22mm (VWR cat no 48366227)
- Cells: 10-14 day cells cultured on Nunc plates
2-3 Nunc 10 Cm (Cat No 172931) dishes labeled with cell
designation, passage and Date
- 2-3 Falcon 10 Cm dishes (3003) with 6 coverslips heat sealed
to bottom of dish and labeled with cell designation, passage and date.
- Remove medium from 1 dish of cells by aspiration, suck dry.
- Add 2 ml of trypsin to cells, roll over surface of plate to cover
- Incubate at 37 or in Laminar Flow hood 3-5 minutes, tap every
minute to knock cells from plate.
- Place 4 ml of medium in a 15 ml conical tube (Sterile)
- Using a 5 ml pipet, pipet trypsin up and down to rinse cells from
surface of dish, when cells are removed, transfer to 15 ml nical tube with
medium. Invert tube to mix.
- Using a Pasteur pipet, remove a sample of cell suspension, load
haemocytometer and count cells. Meanwhile, pellet cells in clinical centrifuge
for 5 min.
- Remove medium from cell pellet, add enough medium to pellet to
give a cell suspension of 5x105 cells/ ml.
- Add 15 ml Complete medium to all dishes. Add 5x105 cells (1 ml)
to dishes with cover slips and 3x105 cells (0.6 ml) to Nunc dishes. If making
extra dishes for biochemical studies, add 5x105 cells (1 ml) to Nunc dishes
containing 15 ml of Complete Medium. Discard left over cells.
- 7% CO2 at 37c
- Monitor integrity of cell culture, evaluate polarity and debris, record
- Feed cells in 10 Cm dishes with 15 ml of Complete Medium on
Monday and Wednesday and 20 ml on Friday.
- Move individual coverslips into 6 Cm dishes 2-3 days after
plating. Feed cells in 6 Cm dishes with 4 ml of Complete Medium Monday,
Wednesday and Friday.