Scrape-loading/dye transfer assay for gap junctional intercellular communication
Solutions needed:
- HBSS + 1% BSA (50 ml; 0.5 g BSA/HBSS)
- PBS + 1% Lucifer Yellow (10 ml for 6 well plates, 0.1 g LY/10 ml PBS)
- PBS
- 2-4% paraformaldehyde
For small cover slips:
Move the cover slip from the 24 well to a 3 cm plate with media. Make sure it’s face up, you can label it with a pen.
- Remove culture medium from a confluent monolayer and save the culture medium in a 10 ml tube. Put it at 37 degrees!
- Rinse cells three times with Hank’s balanced salt solution containing 1% bovine serum albumin (HBC).
- Move the coverslip on parafilm face up.
- Use 30G 1/2 needle to create two longitudinal scratches through the cell monolayer in the presence of a solution of Dulbecco’s phosphate buffered saline containing 100 ul of 0.5% LY and 0.5 % Dextran.
- After exactly 1 min move back the coverslip in the plate and quickly rinse the culture three times with HBC
- Incubate for an additional 8 min (Lucifer yellow) or 2 min (biocytin) in the saved culture medium to allow the loaded dye to transfer to adjoining cells.
- Rinse cells three times with HBSS and fix cells 20 minutes in 4% pfa..
- Rinse cells with PBS 4 times, mount for LM.
- Visualize with fluorescence microscopy.
12/5/2003 G.E.S.
11-2012 Cinzia Ambrosi