Overview
The best transfection reagent that Blaise tried in his plasmid-transfection escapade to collect Dual-Luciferase and fluorescence-reporter data for the AFAR 2021 grant in MEF cells was ViaFect by Promega: https://www.promega.com/products/luciferase-assays/transfection-reagents/viafect-transfection-reagent/?catNum=E4981#protocols.
The other methods attempted included: NEON electroporation, FuGene HD. If ViaFect was not able to be used for some reason, then the next thing that I would try on MEF cells would be FuGene 6 or by using the Amaza Nucleofector in the AIM core. Note that the NEON electroporator was very effective at CRISPR transfection, however, I could not optimize it for plasmids.
Controls available: There is an eGFP-cortactin plasmid frozen down in bacteria in the -80. This could be useful if you have poor transfection efficiencies. The best results that I got were when I would use 1.5-2ug DNA and 12ul Viafect, an effective 1:6 ratio of DNA to the reagent. The cell viability, although not quantified with a Live/Dead stain, is high.
Stable cell line notes: Kanamycin can not be used to generate mammalian cell lines. Neomycin is what Blaise used. In the case of the Dual-luciferase assay, one cannot make a stable cell line and thus, the assay must be done soon after transfection.
Procedure
- Seed cells in a 96 well plate (use a black/clear plate if you are visualizing fluorescence on the EVOS fl auto (cell culture room BRF 221) or the CX7 cellomics (AIM core). Each ViaFect-DNA mixture outlined below can transfect up to 10 wells. (However, if troubleshooting, more or less mixture can be used per each well.)
- Wait for cells to grow to 60-80% confluence.
- * This is VERY IMPORTANT. If you do not wait for the cells to get to this confluence, they will die and transfection will be _very_ low.
- Replace old media with 88ul serum-free and antibiotic-free media.
- Note that serum-free media will kill your cells if you did not let them grow to the required confluence.
- Note that your aseptic technique must adequately be maintained when using antibiotic-free media. Using Antibiotics in this transfection decreases cell viability and transfection efficiency (qualitative observations BLM 4/21/21)
- In a separate, empty well on your 96-well plate, add 100ul serum-free, antibiotic-free media. This will be the basis for your ViaFect-DNA mixture.
- Add 2ug DNA to the mixture.
- Add 12ul ViaFect to the mixture. Pipette 15-20 times to mix.
- Incubate for 15 minutes at room temperature.
- Pipette 10ul of this mixture into each well you are transfecting.
- Incubate in the cell culture incubator for 24-48 hours. If you are trained on the EVOS, you can visualize the transfection over time. The EVOS has a TxRed filter for RPF and a GFP filter.
- When done, visualize with fluorescence to ensure there is transfection or transition into your dual luciferase assay.