Smolke:Protocols/Adherent cell care and feeding: Difference between revisions
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**Wash plate with trypsin (800 ul for 6-cm dish; 3 ml for 10-cm dish) and quickly aspirate away | **Wash plate with trypsin (800 ul for 6-cm dish; 3 ml for 10-cm dish) and quickly aspirate away | ||
**Add another volume of trypsin (same as above) and store in 37C incubator for 10 min | **Add another volume of trypsin (same as above) and store in 37C incubator for 10 min | ||
**Neutralize with media (1 ml for 6-cm dish; 3 ml for 10-cm dish | **Neutralize with media (1 ml for 6-cm dish; 3 ml for 10-cm dish) | ||
**Mix well by pipetting | **Mix well by pipetting | ||
**Sample 10 ul and place in 600-ul microfuge tube | **Sample 10 ul and place in 600-ul microfuge tube | ||
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**Add 12.5 ul diluted cells to 12.5 ul Trypan Blue | **Add 12.5 ul diluted cells to 12.5 ul Trypan Blue | ||
**Count cells with hemocytometer: count 4 quadrants and divide by 20 to get million cells/ml | **Count cells with hemocytometer: count 4 quadrants and divide by 20 to get million cells/ml | ||
**Seed new dish of the appropriate size at 0.02 million cells/ml | **Seed new dish of the appropriate size at 0.02 million cells/ml (make sure cells in the original dish are well resuspended before you take an aliquot to seed the new dish) | ||
Revision as of 20:42, 27 September 2010
Describe the procedures you use when working with adherent cells such as HEK. After everyone has posted contribution we will combine them to create a standard protocol.
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