We have been using the standard calcium phosphate transfection technique for many years with good success. Other labs have successfully used some of the lipid-based transfection reagents, but we have found this method to be the most reproducible and economical.
Cells are grown on non-tissue culture dishes (i.e. petri dishes) and are maintained in RPMI 1640+10% FBS and should never be allowed to be overgrown.
1 Day Prior to Transfection:
Feed cells with fresh media or split cells to be at <1 X 106/ml at time of transfection. These cells double about once a day so this means that you would seed them at about 4 X 105/ml 24 hrs prior to transfection. You will use 7.5 X 106/transfection so plan accordingly. Each transfection will result in 6 60mm dishes so you have triplicate determinations for stimulated and unstimulated with each plasmid.
Day of Transfection:
1. Prepare DNA precipitate:
DNA (12.5-17.5 µg)
2. Harvest cells while DNA
3. Resuspend cells in 1.0 ml of the precipitate; incubate 20 min at RT
4. Add 9 ml complete media to the cells and transfer to 100 mm petri dishes (non-tissue culture type)
5. Incubate 2 hrs at 37o
6. Add chloroquine to 100µM (10 µl of 100mM stock)
7. Incubate 3 hrs at 37o
8. Spray and/or scrape cells off of the dish and spin down in 17X100mm ploypro tubes 5'@ 1000rpm
9. Wash once with serum-free RPMI; spin 5'@ 1000rpm
10. Glycerol shock 90 seconds with 0.5 ml 15% glycerol in PBS; Add 10 ml serum-free RPMI; centrifuge 5' @ 1200 RPM
11. Resuspend cells in 30 ml RPMI + 10% FBS; Plate 5 ml/ 60 mm tissue culture dish and return to incubator
Stimulate as required, usually 8 h with 1 µg/ml LPS
Harvest by scraping into 1ml PBS, spin down in microfuge set on 4 for 5min
Lyse for luciferase assay in 100 m l lysis buffer.
Alternatively, we lyse the cells right on the plate after a PBS wash. Transfer the lysate to an eppendorf tube and centrifuge to pellet debris.
HEPES, pH 7.05
pH to 7.05; sterile filter. Store at RT
2 M CaCl2 Sterile Filter, store at -20
Lysis buffer Make fresh from stocks
KH2PO4, pH 7.8