Sack: Bead Cell Binding

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Current revision (22:08, 10 October 2012) (view source)
 
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*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   
*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
*add beads ASAP, keep incubating at 37 degree water bath
 +
*Return to [[Sack:Protocols]]
*Return to [[Sack:Protocols]]

Current revision

10mm dish cell scraping protocol

Materials:
(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)

Objective

  • 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


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