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Transfection of RAW 264.7 Murine Macrophages

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

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 37o

6. Add chloroquine to 100M (10 l 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

Day 2:

Change media

Day 3:

Stimulate as required, usually 8 h with 1 g/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