1. The day before the transfection trypsinize a highly confluent 10 cm dish of COS cells, take up the cells in 10 ml of DME/10% NuSerum, and add to 50 mls of DME/10% NuSerum in a large square dish. The cells should be approx. 50% confluent at the time of the transfection. 2. For the transfection add 30 micrograms of DNA to each of the dishes, then mix the stack by sloshing around. Add 2.5 ml of DEAE-dextran/Chloroquin to each plate and slosh the stack. Incubate at 37 C in the CO2 incubator for 3-4 hrs. Watch the cells carefully beginning at 2 1/2 hrs. for signs of cell death. DMSO shock before 10% of the cells have died. 3. For DMSO shock, pour the media into a beaker and add 25 mls of PBS, 10% DMSO. Incubate at room temperature for 2 min, pour off or aspirate the PBS/DMSO and replace with 100 mls of DME/1% NuSerum or DME/nutridoma. 4. Four to eight days after the transfection pour the media from the transfected plates into a 250 or 500 ml nalgene bottle and spin in the J6 centrifuge at 4200 rpm for 15 min. Transfer the supernatant to a new bottle, add 1/100 volume of a 2% stock solution of Na azide and store at 4 C. 5. To reuse the large square tissue culture dishes wash with tap water, followed by bleach. Rinse with distilled water and then rinse with 70% ETOH. Allow to dry in the hood with the UV germicidal lamp on for at least 20 min. genetics.mgh.harvard.edu> cat deae.pro DEAE Dextran transfection conditions for hardy cells (e.g. HeLa, COS, CV-1, some Ltk cells), based on Milman and Sussman, Lopata et al., and Seed and Aruffo. For HeLa cells, the concentration of DEAE should be reduced, to 200 ug/ml, and the cells should be carefully monitored, starting at 2 hours after addition of the DEAE/chloroquine. For transfection the DNA, DEAE-dextran and chloroquine can be added in almost any sequence. The only important thing to avoid is addition of concentrated DNA to a solution containing DEAE-dextran, because a precipitate can form. Precipitates usually do not form if the DNA concentration is lower than 1 mg/ml. If a number of different DNA samples are being transfected simultaneously, a convenient stratagem is to add the DNA to the small amount of media remaining after aspirating the old medium just before adding the DME/NuSerum. We typically split the cells to be transfected the night before, to give 50-80% confluence. One simple way to achieve this is to resuspend the trypsinized cells from a confluent 10cm dish in 10ml of medium, and distribute 6 drops from a 10ml pipet into each 6cm dish to be transfected. The higher the degree of confluence, the greater the ability of the cells to withstand the transfection conditions; however expressionfollowing transfection at high density is typically inferior to that at lower density. If the cells are allowed to grow for more than one day they usually are more resistant to the DEAE-dextran, and so better transfections will be obtained if the DEAE and chloroquine concentrations are doubled. Cells are transfected in DME or IMDM containing 10% heat-inactivated NuSerum (Collaborative research). NuSerum allows the cells to withstand the transfection conditions for greater lengths of time, resulting in improved net expression. If Calf or Fetal Bovine Serum is used, a thick precipitate forms in a substantial fraction of transfections, which results in severely eroded cell viability and expression. Presumably the lower protein concentration in the NuSerum obviates this. If the cells were split into DME or IMDM/NuSerum, the medium need not be removed for the transfection. Usually 100 microliters of the DEAE/DNA mix are added to 2ml of medium in a 6cm dish. To further boost expression chloroquine diphosphate can be added to the medium to give a 100 micromolar final concentration. Mazahir Hasan has found that the net expression does not substantially vary with the time of chloroquine addition (i.e. it may be added before, during, or after transfection, as long as the net duration of exposure is controlled to avoid cell death). The optimal time for exposure to the DEAE/chloroquine mixture depends on the state of the cells. In general the best transfections are those that kill 10-20% of the cells; the doomed cells can be easily recognized because their nuclei are shrunken with clear boundaries and often an iridescent appearance by phase contrast microscopy. In general there is a lot of vacuolization of the remaining cells at this stage. As a general guide, 4 hours of exposure to the DEAE/chloroquine will result in the death of about 10% of the cells. At this time the DEAE-containing medium is removed by aspiration, and the plate is covered with 10% DMSO in PBS. The exact quantity is not important, although generally it's hard to cover a 10 cm dish with less than around 3 ml. 6 cm dishes conveniently take 1 ml. After two minutes or longer at room temperature (timing is not important as long as 2 min. minimum is reached), the PBS/DMSO is removed, fresh medium is added, and the cells left to incubate for however long is desired before assay (usually two to three days). If the cells are to be used for an adhesion assay, or receptor binding, etc, it is a good idea to trypsinize the cells within 24 h of DMSO shock, and replate on fresh dishes (without dilution). This makes the cells substantially less sticky.