Sack: Cell Harvesting for Electrophysiology

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(New page: Cell Plating for Electrophysiology<br> Jon Sack Feb 3, 2011<br> Ken Eum April 12, 2011 (Edit)<br> Notes: Steps should be done (not necessary if cell stock is only being used for plating c...)
Current revision (19:10, 15 January 2013) (view source)
 
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Cell Plating for Electrophysiology<br>
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{{Template: Sack Lab}}<br>
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Jon Sack Feb 3, 2011<br>
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<div style="right: center; text-align: left; float:none">
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Ken Eum April 12, 2011 (Edit)<br>
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Notes: Steps should be done (not necessary if cell stock is only being used for plating cells for electrophysiology) inside of a Biological Safety Cabinet in Sterile conditions.
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Cell Harvesting for Electrophysiology<br>
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Ken Eum July 18, 2012 (edit)<br>
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Procedures (in 35mm dish):<br>
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Notes: Steps should be done (not necessary if cell stock is only being used for harvesting cells for electrophysiology) inside of a Biological Safety Cabinet in Sterile conditions.
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# Check under microscope to make sure that cells are less than 80% confluent
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# Carefully remove (aspirate) the solution from the T-25 flask containing the cells
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Procedures (in 35mm dish):
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# Rinse with DPBS (Divalent free Dulbecco’s Phosphate Buffered Saline) ~ 5 mL
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# Check under microscope to make sure that cells are ~80% confluent
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# Remove (aspirate) the DPBS solution
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# Carefully remove (aspirate) the solution from the 35mm dish containing the cells
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# Add 1 mL 0.05% Trypsin EDTA
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#       Rinse with Versene ~ 2 mL
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# Place in 37° C incubator until cells detach from the dish ~ 5 min
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# Remove (aspirate) the Versene solution
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#*Check under the microscope to make sure cells have detached.  If the cells are not yet detached, lightly tap the dish against the table.
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# Add 2 mL of Versene to detach cells
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# Pipet up and down 5-10 times in the Biological Safety Cabinet
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# Wait ~1-2 minutes and scrape off cells with a cell scraper
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#*Check under the microscope to make sure the cells are dispersed into single cells.
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# Transfer the suspended cells into a 15mL conical tube
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# Add 2 mL of Cell Media with selection agents into new 35mm dishes
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# Add 3mL of CHO-SFM – II + 25mM HEPES + 1% Pen/Strep into the 15mL conical tube with the suspended cells.
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# Add 1 drop of the trypsinized cells into each 35mm dish with the Cell Media
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# Spin at 500rcf for 2 minutes
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# Swirl the dish or pipet up and down
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# Aspirate out the supernatant
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# If using Kv2.1 T-Rex CHO cells, add tetracycline (1µg/mL) for 1 hour.  After 1 hour, replace with fresh media or the external solution for electrophysiology recordings.
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# Resuspend with 2mL of CHO-SFM-II + 25mM HEPES
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# Label the Dish: Name, Date, Cell Line, Passage #, Ratio
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# Transfer the suspended cells into a 2mL tube
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*Cell Media: Ham’s – F12 from Gibco, 10% FBS, 1% Penicillin/Streptomycin
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To make add 50 mL Media + 50 mL FBS
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**Cell Media with Selection Agents (Zeocin and Blasticidin)
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F12 from Gibco, 10% FBS, 1% Penicillin/Streptomycin, 1/10,000 Blasticidin, 1/4,000 Zeocin
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Current revision


Sack and Yarov-Yarovoy Labs

Department of Physiology and Membrane Biology
University of California, Davis

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Cell Harvesting for Electrophysiology
Ken Eum July 18, 2012 (edit)

Notes: Steps should be done (not necessary if cell stock is only being used for harvesting cells for electrophysiology) inside of a Biological Safety Cabinet in Sterile conditions.

Procedures (in 35mm dish):

  1. Check under microscope to make sure that cells are ~80% confluent
  2. Carefully remove (aspirate) the solution from the 35mm dish containing the cells
  3. Rinse with Versene ~ 2 mL
  4. Remove (aspirate) the Versene solution
  5. Add 2 mL of Versene to detach cells
  6. Wait ~1-2 minutes and scrape off cells with a cell scraper
  7. Transfer the suspended cells into a 15mL conical tube
  8. Add 3mL of CHO-SFM – II + 25mM HEPES + 1% Pen/Strep into the 15mL conical tube with the suspended cells.
  9. Spin at 500rcf for 2 minutes
  10. Aspirate out the supernatant
  11. Resuspend with 2mL of CHO-SFM-II + 25mM HEPES
  12. Transfer the suspended cells into a 2mL tube
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