Tina:MESC rainbow line: Difference between revisions

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>TinaLo
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>TinaLo
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**Passage feeder free mESCs: P9 to P10. Seed in 1:6 (one well, without MEF).
**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).
**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).




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*'''6/4'''
*'''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.
**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 6/6 to 6/12.
**Cells can be used to produce MEF conditioned medium from 6/7 to 6/13.


==Osteogenic differentiation==
==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]