Tina:MESC rainbow line: Difference between revisions
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**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 (from one 1:12 well and the remaining part of the passaged 1:12 well) | ||
**Prepare 0.1% gelatin | **[[Prepare 0.1% gelatin]] | ||
Add 0.2g gelatin to 200mL ddH2O. Gelatin will not be soluble at this stage. | 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. | Autoclave for 30 min. Gelatin will dissolve. Store at room temperature. | ||
**Prepare gelatin coated plates | **[[Prepare gelatin coated plates]] | ||
Add 1mL of 0.1% gelatin into each well of a six well plate. | Add 1mL of 0.1% gelatin into each well of a six well plate. | ||
Put the plate in 37C incubator for 15-20 min. | 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. | 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) | **Seed MEF on gelatin coating plates (using [[MEF culture medium]]) | ||
*'''5/26''' | *'''5/26''' | ||
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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). | ||
*'''5/30''' | |||
**Change medium of all the P8 InsACGGsAlov3 cells. | |||
*'''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. |
Revision as of 00:01, 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
- 5/8 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.
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
- 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
- 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.