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)            440µl
            2M CaCl2, no need to mix    62µl
            Slowly add 2X HBS             500µl

Incubate 20 min @ RT.

2. Harvest cells while DNA is precipitating

Count viable cells by trypan blue exclusion.
Spin down 7.5 X 10⁶ cells per transfection in 17X100mm polypro tubes

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 37^o

6. Add chloroquine to 100µM (10 µl of 100mM stock)

7. Incubate 3 hrs at 37^o

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

Day 2:

Change media

Day 3:

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.

Reagents:

2X HBS:

    50 mM HEPES, pH 7.05
    1.5 mM Na₂HPO₄
    10 mM KCl
    280 mM NaCl
    12 mM glucose

pH to 7.05; sterile filter.  Store at RT

2 M CaCl₂ Sterile Filter, store at -20

Lysis buffer  Make fresh from stocks

    0.1 M KH2PO4, pH 7.8
    0.1% Triton X-100
    1 mM DTT