Tina:MESC rainbow line: Difference between revisions
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>TinaLo |
>TinaLo mNo edit summary |
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(9 intermediate revisions by the same user not shown) | |||
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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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**P6 cells look nice. Cells were passaged well yesterday. | **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. | **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== | |||
==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
- 5/8 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. 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.