Many cultured cell types do not typically adhere well to glass bottom plates. Adding a thin layer of gelatin can help the cells to adhere better.
Gelatin preparation should be done before transfection is started (before you start seeding the cells).
- Find the 0.1X bottle of gelatin. It is already at the proper concentration. It is usually kept above the TC room centrifuge. Otherwise, possibly check the Cold Room if the TC room was recently cleaned.
- Pipette gelatin into each well to be used of the plate such that the bottom of the well is fully coated with gelatin.
- For a 24 well plate, add 0.5 to 1.0 mL gelatin per well
- For other plates, scale appropriately --> glass 35 mm dishes would be at least 1.5 mL
- More recommendations by Millipore: http://www.millipore.com/userguides/tech1/mcproto045
- Swirl plate (figure eight or shake forward/side) gently to evenly spread the gelatin. (Make sure the bottom of each well is covered!)
- Let gelatin set for 20 minutes. In the meantime, proceed normally with transfection protocol.
- Just prior to seeding the cells, gently aspirate the gelatin from each well. (Make sure you remove all the liquid but don't scrape the bottom of the plate -- tilt the plate to make it easier)
- Trypsinize the cells and neutralize with complete media
- Centrifuge down the cells at 5000 rcf for one minute
- Add 500ul PBS to re-suspend cells
- Immediately proceed with seeding the cells and continue the rest of the transfection protocol.
- Gelatin aliquots can be stored at room temperature.