Previous references said that this solution is prepared fresh by diluting 200 mM probenecid stock solution (Dissolved in 200 mM NaOH) into HBSS Assay Buffer.
Hello
If you used Fluorescent Ca2 Assay for HEK293/Gα16/M2R Cells , yes All HBSS buffer used containing 1mM probenicid, including dye loading buffer, washing buffer and buffer to prepare agonist, etc. This solution is prepared fresh by diluting 200 mM probenecid stock solution (Dissolved in 200 mM NaOH) into HBSS Assay Buffer. follow this protocol
1. Lightly trypsinize the cells and seed them into a 96-well plate (Metrigel coating) at 25,000 cells/well (100 µl per well) in complete culture medium.
2. Culture cells in 5% CO2 at 37°C for 24 hours (at least overnight).
3. Discard the growth media carefully using multi-channel pipette.
4. Wash cells once carefully with 100 µl/well HBSS (containing 1 mM probenecid) using multi-channel pipette.
5. Stain cells with Fluo3/AM. Reconstitute the Fluo3/AM to a concentration of 2.0 mM for the stock solution. Dilute the Fluo3/AM of 2 mM stock to 4 µM in HBSS as the loading solution (containing 1 mM probenecid, 1X red dye). Stain cells with 100 µl/well of the loading solution.
6. Incubate the plate at 37°C in the dark for one hour.
7. Prepare agonist addition plates in advance of assay. To a 96-well plate, add 5X working concentration of carbachol in HBSS, and add 25 µl/well to cell plate.
8. Read with Flexstation using the specified settings and save data.
Good Luck
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