DEAE Dextran Transfection of Mono Mac 6
on day -1
- harvest, spin at 400 g , reseed in completely fresh culture medium either at the same cell density or at a 1:2 to 1:1.5 dilution, you may use the old culture plates (plus fresh ones).
on day 0
- harvest, wash with plain RPMI 1640 + glutamine once, resuspend 10 7 cells in 1 ml plain RPMI 1640 + glutamine, containing DEAE Dextran (50-250 µg/ml final) (Pharmacia 17-0350-1).
- Plasmid DNA is added at 5-10 µg in a volume of about 10 µl into round bottom tissue culture tubes and the DEAE Dextran cell suspension is added on top.*
- incubate 90 min at 37 0 C, resuspend after 30 min and 60 min
- add dimethyl sulfoxide (DMSO), 10% final, resuspend
- wait 2-3 min at RT
- add 9 ml RPMI 1640, spin at 400g
- resuspended in 8 ml of complete medium,
- seed into 4 wells of 24 well plate, incubate at 37 0 C overnight.
A ready to use solution from Promega (#E1210) can also be employed.
on day +1
- For stimulation add stimulant directly to the wells, resuspend 1x
- let the cells go for instance for 4 hs.
- harvest
- test reportergene