Tina:MESC rainbow line
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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. Add 3 ml of mouse ES cell culture medium per well.
- 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 (from one 1:12 well and the remaining part of the passaged 1:12 well)
- Preparation of 0.1% gelatin solution
- Preparation of gelatin coated plates
- Seed MEF on gelatin coating plates (using MEF culture medium)
- 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
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 6/6 to 6/12.