Tina:MESC rainbow line: Difference between revisions

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**Received one vial of cells from Inna, and stored it in the N2(l) tank located at CERC.
**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'''
*'''5/3'''
**discussed with Dr. Blue Lake in Dr. Yang Xu’s lab


*'''5/7 preparing MEF with Blue'''
*'''5/7 preparing MEF with Blue'''


*'''5/8 thawing'''  
*'''5/8 thawing'''  
**Thaw cells in one 6-well plate
**Thaw cells in one 6-well plate. Add 3 ml of [[mouse ES cell culture medium]] per well.


*'''5/11'''
*'''5/11'''
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*'''5/18 Passaging'''  
*'''5/18 Passaging'''  
**Passage the 1:3 well into two 1:3 wells, two 1:6 wells, and one 1:12 well.
**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).
**Frozen down 3 vials of P4 InsACGGsAlov3 in [[mouse ES cell freezing medium]] (from one 1:4 well, one 1:5 well, and one 1:6 well).


*'''5/21 Passaging (Blue and Tina)'''
*'''5/21 Passaging (Blue and Tina)'''
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*'''5/25'''
*'''5/25'''
**Passage one well of 1:12 P6 InsACGGsAlov3 into 1:6 (1 well), 1:12 (1 well) and 1:24 (1 well)
**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)
**Frozen down 3 vials of P6 InsACGGsAlov3. Store at N2(l) tank, Box3, Location 16,17,18.
**[[Preparation of 0.1% gelatin solution]]
**[[Preparation of 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'''
*'''5/28'''
**Passage one well of 1:12 P7 InsACGGsAlov3 mESCs into 1:6 (1 well) and 1:12 (1 well).
**Passage: P7 to P8. Seed in 1:6 (1 well) and 1:12 (1 well).
 
*'''5/30'''
**Change medium of all the P8 InsACGGsAlov3 cells.


*'''5/31'''
*'''5/31'''
**Frozen down 3 vials of P8 InsACGGsAlov3 (from one 1:6 well).
**Frozen down 3 vials of P8 InsACGGsAlov3. Store at N2(l) tank, Box3, Location 19,20,21.  


*'''6/1'''
*'''6/1'''
**Passage one well of 1:12 P8 InsACGGsAlov3 mESCs into 1:6 (1 well) and 1:3 (1 well; on 0.1% gelatin coating plate without MEF feeder cells; add [[MEF conditioned media]])
**Passage: P8 to P9. Seed in 1:6 (1 well, with MEF) and 1:3 (one well, without MEF feeder cells).
  Seed cells without MEF to test the proliferation ability of InsACGGsAlov3 cells under feeder free culture system.
  Seed cells without MEF to test the proliferation ability of InsACGGsAlov3 cells under feeder free culture system.
*'''6/4'''
**Passage feeder free mESCs: P9 to P10. Seed in 1:6 (one well, without MEF).
**Passage: P9 to P10. Seed in 1:6 (one well, with MEF).
*'''6/6'''
**Passage feeder free mESCs: P10 to P11. Seed in 1:12 (one well, without MEF).
==Inactivated MEF cells preparation==
==MEF conditioned medium preparation==
*'''5/29'''
**Start to collect MEF conditioned medium. MEF cells seed on 5/25 were used. Cell density: 2.5*10^4 cells/cm2. 10 wells.
**5/30~6/4 collect MEF conditioned medium every 24 hrs (106 mL in total) from these MEF cells.
*'''5/31'''
**Thaw one vial of MEF (from Dr. Xu's Lab), seed in one T75 flask. Cell density: 4*10^4 cells/cm2. Anticipated production of conditioned medium: 36 mL/day (theoretically 0.3-0.4 mL/cm2).
**Cells can be used to produce MEF conditioned medium from 6/2 to 6/8.
*'''6/4'''
**Thaw one vial of MEF (CF-1 MITC treated, from GlobalStem, Cat# GSC-6001M, Lot# 94300663) in one T75 and 2 wells of one 6-well plate. Cell density :~4*10^4 cells/cm2. Anticipated production of conditioned medium: 40 mL/day.
**Cells can be used to produce MEF conditioned medium from 6/7 to 6/13.
==Osteogenic differentiation==

Latest revision as of 23:40, 6 June 2012

mESC rainbow line expansion[edit]

  • 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
  • 5/11
    • 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 in mouse ES cell freezing medium (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. Store at N2(l) tank, Box3, Location 16,17,18.
  • 5/28
    • Passage: P7 to P8. Seed in 1:6 (1 well) and 1:12 (1 well).
  • 5/31
    • Frozen down 3 vials of P8 InsACGGsAlov3. Store at N2(l) tank, Box3, Location 19,20,21.
  • 6/1
    • Passage: P8 to P9. Seed in 1:6 (1 well, with MEF) and 1:3 (one well, without MEF feeder cells).
Seed cells without MEF to test the proliferation ability of InsACGGsAlov3 cells under feeder free culture system.
  • 6/4
    • Passage feeder free mESCs: P9 to P10. Seed in 1:6 (one well, without MEF).
    • Passage: P9 to P10. Seed in 1:6 (one well, with MEF).
  • 6/6
    • Passage feeder free mESCs: P10 to P11. Seed in 1:12 (one well, without MEF).


Inactivated MEF cells preparation[edit]

MEF conditioned medium preparation[edit]

  • 5/29
    • Start to collect MEF conditioned medium. MEF cells seed on 5/25 were used. Cell density: 2.5*10^4 cells/cm2. 10 wells.
    • 5/30~6/4 collect MEF conditioned medium every 24 hrs (106 mL in total) from these MEF cells.
  • 5/31
    • Thaw one vial of MEF (from Dr. Xu's Lab), seed in one T75 flask. Cell density: 4*10^4 cells/cm2. Anticipated production of conditioned medium: 36 mL/day (theoretically 0.3-0.4 mL/cm2).
    • Cells can be used to produce MEF conditioned medium from 6/2 to 6/8.
  • 6/4
    • Thaw one vial of MEF (CF-1 MITC treated, from GlobalStem, Cat# GSC-6001M, Lot# 94300663) in one T75 and 2 wells of one 6-well plate. Cell density :~4*10^4 cells/cm2. Anticipated production of conditioned medium: 40 mL/day.
    • Cells can be used to produce MEF conditioned medium from 6/7 to 6/13.

Osteogenic differentiation[edit]