Tina:MESC rainbow line: Difference between revisions

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>TinaLo
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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'''
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*'''5/28'''
*'''5/28'''
**Passage one well of 1:12 P7 InsACGGsAlov3 mESCs into 1:6 (1 well) and 1:12 (1 well).
**Passage one well of 1:12 P7 InsACGGsAlov3 mESCs into 1:6 (1 well) and 1:12 (1 well).
*'''5/30'''
**Change medium of all the P8 InsACGGsAlov3 cells.


*'''5/31'''
*'''5/31'''
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**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 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]])
  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.
==Inactivated MEF cells preparation==
*'''5/31'''
**Received one vial of untreated CF-1 MEF cells. (GlobalStem; cat# GSC-6001M; 4*10^6 cells/vial)
==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'''
**Collect 20 mL MEF conditioned medium. repeat the same protocol to collect more conditioned medium.
*'''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: 0.3-0.4 mL/cm2.
==Osteogenic differentiation==

Revision as of 01:51, 2 June 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
  • 5/11
    • 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)
  • 5/26
    • Change medium of all the P7 InsACGGsAlov3 cells.
  • 5/28
    • Passage one well of 1:12 P7 InsACGGsAlov3 mESCs into 1:6 (1 well) and 1:12 (1 well).
  • 5/31
    • Frozen down 3 vials of P8 InsACGGsAlov3 (from one 1:6 well).
  • 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)
Seed cells without MEF to test the proliferation ability of InsACGGsAlov3 cells under feeder free culture system.


Inactivated MEF cells preparation

  • 5/31
    • Received one vial of untreated CF-1 MEF cells. (GlobalStem; cat# GSC-6001M; 4*10^6 cells/vial)


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
    • Collect 20 mL MEF conditioned medium. repeat the same protocol to collect more conditioned medium.
  • 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: 0.3-0.4 mL/cm2.


Osteogenic differentiation