Smolke:Protocols/Adherent cell care and feeding: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 23: | Line 23: | ||
'''Yvonne''' | '''Yvonne''' | ||
*General comments: | *General comments: | ||
*Changing media - | **All comments here refer specifically to HEK cells. | ||
*Subculturing - | **Volumes | ||
***24-well plate: 0.5 ml/well | |||
***6-cm dish: 3 ml total | |||
***10-cm dish: 10 ml total | |||
***I was taught to use these volumes by Steph/Chase. However, please note that they don't actually scale with the growth areas of the dishes (2 cm^2 for 24-well plate, 21 cm^2 for 6-cm dish, and 58 cm^2 for 10-cm dish). Therefore, plates/dishes seeded at the same density in terms of million cells/ml will not have the same confluency. | |||
**General techniques | |||
***Lift lid and tilt plate with one hand while aspirating/pipetting with the other | |||
***The lid should cover the plate as much as possible, providing just enough opening to perform the required task | |||
***The lid opening should face away from the hood opening (i.e., away from you and toward the back wall) | |||
***Tilt the plate toward the back when aspirating | |||
***Eject media gently against the side wall of the dish to avoid dislodging cells | |||
***When aspirating, immerse the pipet tip in the liquid but don't touch the plate until the very end to avoid aspirating away cells | |||
**As with all cell culturing, it is desirable to be as consistent as possible in general maintenance. Therefore, I always count the cells when I subculture and seed at the same density each time (unless I have specific reasons to seed at a different density). I also feed the cells on a regular schedule. | |||
*Changing media | |||
***Aspirate away old media and add fresh media as described above; add antibiotics (for selection pressure) as necessary | |||
***If seeding 6-cm dish at 0.02 million cells/ml (see below), feed cells 3 and 5 days after seeding; cells should be ready for subculturing on day 6 | |||
*Subculturing | |||
**Aspirate away old media | |||
**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 | |||
**Neutralize with media (1 ml for 6-cm dish; 3 ml for 10-cm dish | |||
**Mix well by pipetting | |||
**Sample 10 ul and place in 600-ul microfuge tube | |||
**Dilute with 90 ul media (make sure it's well mixed) | |||
**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 | |||
**Seed new dish of the appropriate size at 0.02 million cells/ml | |||
Revision as of 20:37, 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.
|