Difference between revisions of "Sack: Bead Cell Binding"
Latest revision as of 19:08, 10 October 2012
10mm dish cell scraping protocol
(2x 10mm dish)
warm 5 ml EDTA DPBS (such as versene) for each plate
two 15 ml falcon tubes
(tube 1: saved media, tube2: scraped cells)
- scrape two 70-90% confluent 10 cm dishes of Kv2.1 TREX cells incubated with1 ug/ml tetracycline for 24-48 hours (1 dish for every 2 tubes to be incubated with beads)
- Perform the same procedure for 2x 10cm dishes of Empty CHO cells.
- to re-suspend a dense amount of cells to plate with beads 5 and 180 K solution after appropriate incubation time
Procedure (for 2x 10cm dishes of a given cell type)
- remove cell media from dishes and save in 15 ml tube
- rinse cell plate with 5mL of EDTA DPBS, aspirate majority of solution leaving a thin layer of EDTA DPBS on cells
- scrape entire plate surface area of cells
- place all of scraped cells in a separate 15mL falcon tube (2)
- rinse dishes with 5mL of their original cell media from tube (1), and add to cell mix
- centrifuge tubes at 500g for 5 minutes; cells will pellet at bottom
- remove supernatant from cell mix, careful not to remove cell pellet formed on bottom.
- re-suspend cells in 4 ml of the supernatant media (tube 2) you've just spun down, triturate 10X to break up cell clumps, and transfer to 2x 2mL tubes
- add beads ASAP, keep incubating at 37 degree water bath
- Return to Sack:Protocols