Page 6 - Cell Culture Training Hand Book - A4 Edited 20.1.2020
P. 6
CULTURE MEDIA PREPARATION (for HEPG2 cells)
Culture Media Recipe (50 ml)
Content
No. Items Concentration Volume (ml) Prepare today
Dulbecco’s Modified Eagle Medium –
1 89% 44.5
DMEM (Low Glucose)
2 FBS (Fetal Bovine Serum) 10% 5
Antibiotic Pen-Strep (Pennicilin-
3 1% 0.5
streptomycin)
Total Volume (ml): 50.0
CELL THAWING AND REVIVE
Reagents
+2
+2
1. DPBS 1x (Dulbecco’s Phosphate Buffered Saline) (- Ca - Mg )
2. Culture media (DMEM with 10% FBS)
Protocol
1. Remove cryo vial from the liquid nitrogen tank.
2. Thaw the vial in 37˚C water bath until completely thawed.
3. Add 6 ml of culture media into 15 ml fresh falcon tube. Then, transfer the cells from the vial
into the falcon tube and suspend the mixture.
4. Centrifuge the cell suspension at 1,500 rpm for 5 minutes. Remove the supernatant. Tab
tube gently to loosen pellet.
5. Remove the supernatant. Tap tube gently to loosen pellet and resuspend the pellet (at
least 50 x, and avoid bubbles ) with 2 ml of culture media.
6. Transfer 3 ml of culture media into fresh culture flask. Then, seed the cells into T25 flask.
7. Spread the cells evenly .
8. Incubate the cells in 5% CO 2 incubator at 37 °C . Change media every 2-3 days .
Tips
1. Adherent cells produce integrins (proteins) that aid attachment to the plate and each other. Calcium and magnesium are
essential to these protein activities (cell binding and clumping). Formulations without these ions are required for washing and
rinsing suspended cells.
2. Constant gentle shaking for one minute will help cell thawed quicker.
3. Usually a few times. Depending on cell type, some need longer time to dissociate cells.
4. Depending on cell type, 1,300-1,500 rpm for 3-5 mins is a common practise.
5. HepG2 cells are commonly grow in clumps, need longer time to dissociate cells.
6. Cells are likely to be trapped in bubbles, avoid bubbles to prevent cell loss when transfering cells into fresh flask.
7. Gently move the flask east-west, north-south to ensure even cell distribution.
8. 4-10% CO 2 is common for most cell culture experiments. Most human and mammalian cell lines are maintained at 36°C to 37°C
for optimal growth.
9. Media change is required to replenish nutrients and keep correct pH of culture. If the media turns orange rather than pinky
orange, it need to be changed as soon as possible.
Cell Culture Training Hand Book | CMBL Page 6 of 12
