Materials
- DMEM high glucose
- Peniciline/streptomycine (100x)
- FBS
- Tryple
- 2% Gelatine
- PBS
Media preparation
- DMEM high glucose
- 1% peniciline/streptomycine
- 10% FBS
Dish coating
- Dilute 2% stock of Gelatine to 0.1% in PBS
- Sterile filter
- Incubate 30 min. at 37◦C
- Aspirate gelatine from the plates
Cells Thawing
- Transfer cells from liquid nitrogen storage on dry ice into cell culture room
- Spray with ethanol, immerse in water bath
- Let thawing for 1-2 min. (check visually, remove when there is only a little piece of ice left inside the vial)
- Transfer content of the vial into a 15mL tube
- Dropwise add cold culture medium, shake gently between the media addition (first 5 ml)
- Add 5 more mL to reach 10-12 ml
- Colllect cells by centrifugation at 300g for 5 minutes
- Discard the supernatant into waste
- Flick the tube to release the cell pellet
Cells counting
- Resuspend in 1mL of media
- take 10uL of susupension, mix with 10uL of trypan blue, inject into the counting slide
- count cells
Cells seeding
- recomended seeding density is 2x104 cells/ cm2
- dilute cells to proper concentration, gently pipette onto the plate
Cell Passaging
- Prepare fresh dishes with gelatine coating (-30 min.)
- Aspirate the media
- Wash with PBS
- Add Tryple (1mL per 20cm2)
- Incubate for 5 min. at 37◦C
- Visually check the cells detachment
- Gently collect using P1000 pipette tip
- Collect to 10mL of medium
- Colllect cells by centrifugation at 300g for 5 minutes
- Discard the supernatant into waste
- Flick the tube to release the cell pellet
- Resuspend in 1 ml of medium
- Count cells
- Resuspend in fresh media to desired concentration • Seed onto the dishes
Recomended cell densities
Sparse cells
5 000 -20 000 cells/cm2
Dense cells
> 50 000 cells/cm2