LX-2 cell culture

Reviving and culture LX2 line from frozen stock

Materials:
Cell culture Media: DMEM (Gibco 11965-092) with 10% FBS and 1% P/S
TrypLE Express (Gibco# 12605-010)
DPBS (1X) without Calcium Chloride and Magnesium Chloride (Gibco#14190-144)
T-75 flasks

Methods:
1. Quick thaw the sample tube (from liquid nitrogen frozen stock) at 37C water bath
2. Once thaw immediately wipe the outer wall of the tube with ethanol
3. Place the tube inside the cell culture hood
4. Mix up the cells very gently by pipetting up and down
5. Transfer the cell suspension into a sterile 15ml centrifuge tube
6. Add 1ml cell culture media to the sample tube, wash shortly and transfer the media in same 15ml tube
7. Add another 8ml of cell culture media in the 15 ml of tube (now total volume 10ml)
8. Centrifuge the tube at 1200rpm for 5 minutes at 4°C (cold centrifuge instrument)
9. Discard the supernatant very carefully and save the cell pellets
10. Immediately add 2ml of cell culture media and resuspend the cell pellets very gently
11. Add 10 ml of additional cell culture media and mix
12. Transfer this whole preparation to one T-75 flask
13. Keep the flask in 37°C 5%CO₂ incubator
14. Next day take out old media and add 10ml of fresh media into to the flask
15. Change cell culture media once a week.

Passaging cell lines (passage the cells only when they are 80-90% confluent):
1. Discard old media from the flask
2. Wash the cells two times with warm (37°C) 1X PBS
3. Add 700μl of warm TrypLE Express (37°C) in the flask (in case of T-75 flask)
4. Keep the flask in the 37°C 5%CO₂ for a minute (critical)
5. Tap the flask by hand several times to loosening the cells from the flask
6. Immediately add 5ml (may add 3 times more than TrypLE Express) of cell culture media
7. Unclamp the cells by vigorously pipetting up and down several times (at least 10 times) inside the flask and check under microscope
8. Transfer the cells to a 15ml centrifuge tube
9. Wash the flask with another 5ml of cell culture media
10. Transfer it to same 15 ml tube
11. Spin the tube with cells at 1200rpm for 5 minutes
12. Discard the supernatant very carefully and save the cell pellets
13. Add 2ml of cell culture media and re-suspend the cell pellets very gently
14. Add another 8ml of cell culture media in it
15. Seed the cells 1:2 in new T-75 flasks
16. Keep the flask in 37°C 5%CO₂ incubator and change the media once a week

Freezing cells:
Making Freezing Media:
DMEM (no FBS or P/S): 7ml (final conc. 70%)
FBS:                                2ml (final conc. 20%)
DMSO:                           1ml (final conc. 10%)
Vortex the freezing media and keep always in ice
1. Discard old media from the flask and wash the cells two times with warm (37°C) 1X PBS
2. Add 700μl of warm TrypLE Express (37°C) in the flask (in case of T-75 flask)
3. Keep the flask in the 37°C 5% CO₂ for a minute (critical)
4. Tap the flask by hand several times to loosening the cells from the flask
5. Immediately add 5ml (may add 3times more than TrypLE Express) of cell culture media
6. Unclamp the cells by vigorously pipetting up and down several times (at least 10 times) inside the flask and check under microscope
7. Transfer the cells to a 15ml centrifuge tube
8. Wash the flask with another 5ml of cell culture media
9. Transfer it to same 15 ml tube
10. Spin the tube with cells at 1200rpm for 5 minutes
11. Discard the supernatant very carefully and save the cell pellets
12. Add about 10-20ml (depending on cell confluency) of cold freezing media and resuspend the cell pellets very gently
13. Count the cell number and mark it in each cryovial (Simport #T309-2A -2ml)
14. Aliquot 1ml cells in each cryovial and keep it in True North Chilling Jar (Heathrow Scientific #HS23230A)
15. Keep the Chilling Jar at -80°C for overnight for slow freezing
16. Next day store the cryovials in liquid nitrogen