Tina:MESC rainbow line: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>TinaLo
>TinaLo
Line 30: Line 30:
**P6 cells look nice. Cells were passaged well yesterday.
**P6 cells look nice. Cells were passaged well yesterday.
**P5 1:12 well shows a few differentiated colonies, and therefore is discarded to maintain good cell quality.
**P5 1:12 well shows a few differentiated colonies, and therefore is discarded to maintain good cell quality.
*'''5/24 Passaging'''
**Bring mESCs back to CERC. Will start to culture by myself.
**Bring back 8 vials of P5 InsACGGsAlov3 and store them in the N2(l) tank located at CERC.
**Passage one well of 1:6 P6 InsACGGsAlov3 into 1:6 (1 well), 1:12 (1 well) and 1:24 (1 well)
**Frozen down 7 vials of P6 InsACGGsAlov3 (from two 1:3 well and one 1:6 well)
*'''5/25'''
**Passage one well of 1:12 P6 InsACGGsAlov3 into 1:6 (1 well), 1:12 (1 well) and 1:24 (1 well)
**Frozen down 3 vials of P6 InsACGGsAlov3 (from one 1:12 well and the remaining part of the passaged 1:12 well)
**Prepare 0.1% gelatin
Add 0.2g gelatin to 200mL ddH2O. Gelatin will not be soluble at this stage.
Autoclave for 30 min. Gelatin will dissolve. Store at room temperature.
**Prepare gelatin coated plates
Add 1mL of 0.1% gelatin into each well of a six well plate.
Put the plate in 37C incubator for 15-20 min.
Plates can be stored in incubator (as long as they are not dry out). Aspirate remaining gelatin before use.
**Seed MEF on gelatin coating plates (using MEF culture medium)
*'''5/26'''
**Change medium of all the P7 InsACGGsAlov3 cells.
*'''5/28'''
**

Revision as of 19:18, 29 May 2012

mESC rainbow line expansion

  • 5/2
    • Received one vial of cells from Inna, and stored it in the N2(l) tank located at CERC.
  • 5/3 discussed with Dr. Blue Lake in Dr. Yang Xu’s lab
  • 5/7 preparing MEF with Blue
  • 5/8 thawing
    • Thaw cells in one 6-well plate
  • 5/11
    • some colonies appear to show
  • 5/14 Passaging
    • The culture looks ok, but there is some differentiation… so Blue passaged at several different dilutions (the lower ones to try and get rid of the differentiated cells).
    • Passage one 6 well into one 1:3, one 1:4, one 1:5, and one 1:6 well.
  • 5/18 Passaging
    • Passage the 1:3 well into two 1:3 wells, two 1:6 wells, and one 1:12 well.
    • Frozen down 3 vials of P4 InsACGGsAlov3 (from one 1:4 well, one 1:5 well, and one 1:6 well).
  • 5/21 Passaging (Blue and Tina)
    • 1:3 wells look very healthy; 1:6 wells look good, and 1:12 well can wait for one more day.
    • Passage one well of 1:3 into 1:3 (1 well), 1:6 (1 well) and 1:12 (1 well)
    • Passage one well of 1:6 into 1:3 (1 well), 1:6 (1 well) and 1:12 (1 well)
    • Frozen down 8 vials of P5 InsACGGsAlov3 (from one 1:3 well and one 1:6 well)
  • 5/22
    • P6 cells look nice. Cells were passaged well yesterday.
    • P5 1:12 well shows a few differentiated colonies, and therefore is discarded to maintain good cell quality.
  • 5/24 Passaging
    • Bring mESCs back to CERC. Will start to culture by myself.
    • Bring back 8 vials of P5 InsACGGsAlov3 and store them in the N2(l) tank located at CERC.
    • Passage one well of 1:6 P6 InsACGGsAlov3 into 1:6 (1 well), 1:12 (1 well) and 1:24 (1 well)
    • Frozen down 7 vials of P6 InsACGGsAlov3 (from two 1:3 well and one 1:6 well)
  • 5/25
    • Passage one well of 1:12 P6 InsACGGsAlov3 into 1:6 (1 well), 1:12 (1 well) and 1:24 (1 well)
    • Frozen down 3 vials of P6 InsACGGsAlov3 (from one 1:12 well and the remaining part of the passaged 1:12 well)
    • Prepare 0.1% gelatin
Add 0.2g gelatin to 200mL ddH2O. Gelatin will not be soluble at this stage.
Autoclave for 30 min. Gelatin will dissolve. Store at room temperature.
    • Prepare gelatin coated plates
Add 1mL of 0.1% gelatin into each well of a six well plate.
Put the plate in 37C incubator for 15-20 min.
Plates can be stored in incubator (as long as they are not dry out). Aspirate remaining gelatin before use.
    • Seed MEF on gelatin coating plates (using MEF culture medium)
  • 5/26
    • Change medium of all the P7 InsACGGsAlov3 cells.
  • 5/28