J774 Cell Transfection
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Protocol
Cell Culture
- Prepare a 12-well plate by adding 1 mL of fresh DMEM per well. If coverslips are required, add one coverslip per well prior to adding media.
- Wash the cells pre-cultured in a T25 flask with 5 ml of PBS, then remove the PBS. Add 1 mL of trypsin for 5 min at 37oC. Add 4 mL of fresh media to deactivate the trypsin, then determine the cell concentration using a hemocytometer.
- Add the cells to the 12-well plate according to the following concentration:
- 500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips => ~35-40 cells/field-of-view at 63x, with ~20% transfected
- 1 million cells/well for subsequent parachuting of cells onto coverslips
- Alternatively, if cells are 80-90% confluent, add 3-5 drops of trypsinzed cells into each well
- Rock the plate sideways to evenly distribute the cells.
- Culture the cells overnight at 37oC.
Transfection
- Thaw the DNA to be used for transfection.
- Set up a reaction for each well in a microcentrifuge tube:
- Add 3.3 µg of DNA to 150 µL of serum-free media. Vortex the tube briefly to mix.
- Add 10 µL of FuGENE-HD Transfection Reagent. Flick the tube 15-20 times to mix. Note: The transfection reagent is ethanol-based and evaporates easily. Keep it in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use.
- Let the reaction sit for 15 min at room temperature in the tissue culture hood, allowing the DNA-containing micelles to form. Note: Do not exceed 20 min of incubation time.
- Add dropwise the total reaction volume of each microcentrifuge tube to its respective cell well. Rock the plate sideways to mix.
- Incubate the transfecting cells 24-48 hr at 37oC.
Paraformaldehyde Cell Fixation
- Aspirate the media. Wash the cells twice with non-sterile PBS, at 1 mL/well. Note: Be careful not to knock the cells off the coverslips during the wash steps.
- Add 1 mL of 4% PFA (diluted in PBS) into each well. Incubate on the plate shaker for 15 min at room temperature in the dark.
- Remove PFA and wash cells with 1 mL/well of non-sterile PBS for 5 min.
- Cover cells with 1 ml PBS/well and keep plate at 4oC.