Growing Cells

From Genome@Yale

Grow IMR90 Cells

1. Cells were cultured according to the directions provided by ATCC (catalog # CCL-186) at 37°C with 5% CO2.
2. Cells were grown in MEM with Earle’s Salts (Invitrogen catalog# 11095-080) supplemented with 10% fetal calf serum (FCS).
3. Cells were maintained in six 175cm2 plates (Corning P/N 431085) with 30mL media each and grown until confluent.
4. Cells were washed with 1X PBS and detached by treatment with 2mL 1X Trypsin/EDTA solution (Invitrogen catalog # 25300-054).
5. Trypsin treated cells were diluted with media with 10% FCS to quench trypsin.
6. Cells were then cultured in 16X 500cm2 plates (Corning P/N 431110) with 100mL media with 10% FCS for chromatin isolation.
7. They were grown until almost confluent (~5 X 107 cells/plate; 16 plates ~5 X 108 cells).

Harvest and Crosslink Cells

1. When cells are close to confluent, aspirate (or pour off) media from each 500cm2 plate. Wash cells once with 100mL 1X PBS.
2. Add 4mL 0.5X Trypsin/EDTA in PBS to each plate.
3. Harvest cells with cell scraper.
4. Pool cells into 250mL conical bottle and bring the volume to 250mL with PBS.
5. Centrifuge 3K rpm 15minutes at 4°C.
6. Resuspend the cell pellet in 200mL PBS (disperse well by repeated pipeting).
7. Add 20mL freshly prepared crosslinking buffer (11% formaldehyde, 0.1M NaCl, 1mM EDTA, 0.5mM EGTA, 50mM Hepes pH 8.0).
8. Incubate 15 minutes at room temperature with rocking.
9. Add 10.5mL 2.5M Glycine to quench crosslinking. Swirl to get it well mixed. 5 minutes at room temperature.
10. Centrifuge 3K rpm 15minutes at 4°C.
11. Resuspend in 25mL PBS. Transfer to a 50mL conical tube.
12. Centrifuge 3K rpm 15 minutes at 4°C and remove supernatant.
13. Freeze and store cell pellets at -80°C until chromatin extraction.

Final                Stock     25mL        50mL
0.1M NaCl            5M        0.5mL       1ml
1mM EDTA             0.5M      50uL        100uL 
0.5mM EGTA           0.5M      25uL        50uL
50mM Hepes pH7.9     1M        1.25mL      2.5mL
dH2O                           15.7mL      31.4mL

Add just prior to use

11% formaldehyde     37%       7.45mL      14.9mL