Growing/splitting E14 ES cells


Posted by cpark
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Protocol
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: 
  1. Prepare gelatin-coated plates by incubating plates with 0.1% gelatin in a 37C incubator for 1~2 hours.
  2. Change media 2-4 hours prior to splitting. Cells must be 60-80% confluent and healthy.
  3. 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. 
  4. After aspirating PBS, add 2ml of pre-warmed trypsin. Be sure to cover the entire plate by carefully swirling it.
  5. 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. 
  6. While cells are incubating, remove gelatin from the pre-treated plates and add 5ml of warmed medium. 
  7. 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.
  8.  Transfer cell suspension to new plates, so the final confluence is between 20-30%, depending on your needs. Do not split below 20% confluency.