Materials
Materials
Passaging/Splitting Guide
|
Plate |
Gelatin/PBS |
Trypsin |
Media (add to inactivate trypsin) |
Media (daily feeding) |
|
96-well |
100ul |
25ul |
175ul |
200ul |
|
24-well |
400ul |
100ul |
700ul |
800ul |
|
6-well |
2ml |
500ul |
2.5ml |
2-3ml |
|
10cm plate |
5ml |
2ml |
8ml |
10ml |
E14 Medium
|
Glasgow Minimum Essential Medium |
Sigma |
G5154 |
404.5ml |
|
FBS (single lot tested) |
Hyclone |
SV30014 |
75ml |
|
Glutamin-I, 100X |
Gibco |
35050-061 |
5ml |
|
Non-essential amino acids, 100X |
|
|
5ml |
|
Pen-Strep, 100X |
|
|
5ml |
|
Sodium pyruvate, 100X |
|
|
5ml |
|
2 (beta)mercaptoethanol *fresh aliquots (1000X stock = 70ul in 10ml PBS w/o Ca or Mg) |
Sigma |
M-7522 |
500ul |
|
LIF (Esgro), 107U/ml |
Gibco |
13275-029 |
50ul |
Methods
Methods
- Prepare gelatin-coated plates by incubating plates with 0.1% gelatin in a 37C incubator for 1~2 hours.
- Change media 2-4 hours prior to splitting. Cells must be 60-80% confluent and healthy.
- When cells are ready to be passaged, aspirate off old media and wash with 10ml pre-warmed PBS. Add PBS gently to the side of the plate to avoid disrupting the cells.
- After aspirating PBS, add 2ml of pre-warmed trypsin. Be sure to cover the entire plate by carefully swirling it.
- Incubate in a 37C incubator for 5 minutes and check under the microscope. If you see big clumps of cells, incubate additional 2-5 minutes. Do not over-trypsinize.
- While cells are incubating, remove gelatin from the pre-treated plates and add 5ml of warmed medium.
- Once majority of the cells are uniformly dispersed into small clumps or single cells, inactivate trypsin by adding 8.5ml of medium. Gently pipette up and down using 10ml pipette 10-15 times without making bubbles.
- Transfer cell suspension to new plates, so the final confluence is between 20-30%, depending on your needs. Do not split below 20% confluency.
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