Tina:Osteogenic differentiation on mES brainbow cells (OSG): Difference between revisions
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== | ==Seeding density test== | ||
*'''Purpose:''' To | *'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation | ||
*''' | *'''Test #1:''' | ||
** | **Date: 7/5/2012 ~ 7/7/2012 | ||
** | **Cells: P11 InsCAGGsAlov3 (mES brainbow cells) | ||
*''' | **7/5: P10 mES brainbow cells (on feeder) were trypsinized. Cells were then divided into two groups. Group one were seeded 1:2, 1:4, 1:6, 1:8 and 1:12 on gelatin coated glass-well immediately. Group two were let sit for 15 min and then seeded 1:2, 1:4, and 1:6 on gelatin coated glass-well. | ||
**Date: | **7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density. | ||
**Cells: | **According to results, seeding at 1:6 with 15 min sitting time is the best seeding density for osteogenic induction (one day after seeding). | ||
** | |||
==Osteogenic differentiation media composition test== | |||
*'''Purpose:''' To test the effects of osteogenic differentiation media on mESCs | |||
*'''Test #1:''' | |||
**Date: 7/1/2012 ~ 7/9/2012 | |||
**Cells: P10 InsCAGGsAlov3 (mES brainbow cells) | |||
**Osteogenic differentiation media (with 1% serum): | |||
{| {{table}} border=1 | {| {{table}} border=1 | ||
| align="center" style="background:#f0f0f0;"|''Component'''' | | align="center" style="background:#f0f0f0;"|''Component'''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Volumn''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Final concentration''' | ||
|- | |- | ||
| | | MEM ||193ml|| | ||
|- | |- | ||
| | | Serum ||2ml||1% | ||
|- | |- | ||
| | | Dexamethasone ||200ul of 0.1mM stock||0.1uM | ||
|- | |- | ||
| | | Ascorbic acid ||1ml of 10mg/ml stock ||50ug/ml | ||
|- | |- | ||
| style="background:#f0f0f0;"| | | b-GP ||2ml of 1M stock||10mM | ||
| style="background:#f0f0f0;"| | |- | ||
| style="background:#f0f0f0;"| | | PenStrep ||2ml||1% | ||
|- | |||
| style="background:#f0f0f0;"|Total | |||
| style="background:#f0f0f0;"|200 mL | |||
| style="background:#f0f0f0;"| | |||
|} | |} | ||
** | **7/1: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 10 min, and then seeded 1:5 (two wells) and 1:10 (4 wells) onto feeder free system (now become P10). | ||
**7/2 and 7/3: Cell density was still low. Induction should wait for one more day. | |||
**7/4: Cell density became too high. Several colonies were formed. Therefore, the best time point to induce osteogenic differentiation is ‘’1-2 days after seeding’’’ (need further confirmation). | |||
**Add induction media to each well on 7/4 to observe the cell morphology changes at the beginning of osteogenic differentiation. | |||
**7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.) | |||
**7/6: Possible cause of cell death: (1)chemical concentration is too high (toxic). (2)serum concentration is too low. | |||
** | Further tests should be arranged (with lower chemical concentration and/or higher serum concentration). | ||
** | |||
** | |||
** | |||
Pictures | |||
** | |||
(1) | |||
( | |||
( | |||
*'''Test #2:''' | *'''Test #2:''' | ||
**Date: 7/ | **Date: 7/13/2012 ~ ongoing work | ||
**Cells: P10 mES brainbow cells. | **Cells: P10 InsCAGGsAlov3 (mES brainbow cells) | ||
**Osteogenic differentiation media: | |||
Basal media were changed from MEM into DMEM. Different concentration of serum were tested. | |||
(1 | {| {{table}} border=1 | ||
| align="center" style="background:#f0f0f0;"|''Component (volumn)/group'''' | |||
| align="center" style="background:#f0f0f0;"|''5% serum'''' | |||
| align="center" style="background:#f0f0f0;"|''15% serum'''' | |||
| align="center" style="background:#f0f0f0;"|''2me'''' | |||
|- | |||
( | | DMEM ||193ml||193ml||193ml | ||
|- | |||
( | | Serum ||5%(10ml)||15%(30ml)||15%(30ml) | ||
|- | |||
( | | 0.1 uM Dexamethasone ||200ul of 0.1mM stock||200ul of 0.1mM stock||200ul of 0.1mM stock | ||
|- | |||
** | | 50ug/ml Ascorbic acid ||1ml of 10mg/ml stock||1ml of 10mg/ml stock ||1ml of 10mg/ml stock | ||
|- | |||
| 10mM b-GP ||2ml of 1M stock||2ml of 1M stock||2ml of 1M stock | |||
|- | |||
| 1% PenStrep ||2ml||2ml||2ml | |||
|- | |||
| 1% Glutamax ||-||-||2ml | |||
|- | |||
| 1% NEAA ||-||-||2ml | |||
|- | |||
| 0.1% 2me ||-||-||200ul | |||
|- | |||
| style="background:#f0f0f0;"|Total | |||
| style="background:#f0f0f0;"|200 mL | |||
| style="background:#f0f0f0;"|200 mL | |||
| style="background:#f0f0f0;"|200 mL | |||
|} | |||
**7/13: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 15 min, and then seeded 1:6 (three wells) onto feeder free system (now become P10). | |||
**7/14(Day 0): PBS wash twice and then add induction media to each well. | |||
**7/15-7/16: Take pictures. Cells look ok. Scheduled to change media three days after induction. | |||
**7/17 (Day 3): Take pictures. Observed some cell death. Change media. | |||
**7/18: Take pictures. Number of cell death were under control. Important finding: Cell death occurred less in 5% serum group. This might suggests that cell death may be due to lack of nutrition. Managed to change induction media daily hereafter. | |||
**7/19-7/23: Change media and take pictures daily. |
Revision as of 18:44, 23 July 2012
Seeding density test
- Purpose: To determine the seeding density of mESCs for osteogenic differentiation
- Test #1:
- Date: 7/5/2012 ~ 7/7/2012
- Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
- 7/5: P10 mES brainbow cells (on feeder) were trypsinized. Cells were then divided into two groups. Group one were seeded 1:2, 1:4, 1:6, 1:8 and 1:12 on gelatin coated glass-well immediately. Group two were let sit for 15 min and then seeded 1:2, 1:4, and 1:6 on gelatin coated glass-well.
- 7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.
- According to results, seeding at 1:6 with 15 min sitting time is the best seeding density for osteogenic induction (one day after seeding).
Osteogenic differentiation media composition test
- Purpose: To test the effects of osteogenic differentiation media on mESCs
- Test #1:
- Date: 7/1/2012 ~ 7/9/2012
- Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media (with 1% serum):
Component'' | Volumn | Final concentration |
MEM | 193ml | |
Serum | 2ml | 1% |
Dexamethasone | 200ul of 0.1mM stock | 0.1uM |
Ascorbic acid | 1ml of 10mg/ml stock | 50ug/ml |
b-GP | 2ml of 1M stock | 10mM |
PenStrep | 2ml | 1% |
Total | 200 mL |
- 7/1: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 10 min, and then seeded 1:5 (two wells) and 1:10 (4 wells) onto feeder free system (now become P10).
- 7/2 and 7/3: Cell density was still low. Induction should wait for one more day.
- 7/4: Cell density became too high. Several colonies were formed. Therefore, the best time point to induce osteogenic differentiation is ‘’1-2 days after seeding’’’ (need further confirmation).
- Add induction media to each well on 7/4 to observe the cell morphology changes at the beginning of osteogenic differentiation.
- 7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.)
- 7/6: Possible cause of cell death: (1)chemical concentration is too high (toxic). (2)serum concentration is too low.
Further tests should be arranged (with lower chemical concentration and/or higher serum concentration).
- Test #2:
- Date: 7/13/2012 ~ ongoing work
- Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media:
Basal media were changed from MEM into DMEM. Different concentration of serum were tested.
Component (volumn)/group'' | 5% serum'' | 15% serum'' | 2me'' |
DMEM | 193ml | 193ml | 193ml |
Serum | 5%(10ml) | 15%(30ml) | 15%(30ml) |
0.1 uM Dexamethasone | 200ul of 0.1mM stock | 200ul of 0.1mM stock | 200ul of 0.1mM stock |
50ug/ml Ascorbic acid | 1ml of 10mg/ml stock | 1ml of 10mg/ml stock | 1ml of 10mg/ml stock |
10mM b-GP | 2ml of 1M stock | 2ml of 1M stock | 2ml of 1M stock |
1% PenStrep | 2ml | 2ml | 2ml |
1% Glutamax | - | - | 2ml |
1% NEAA | - | - | 2ml |
0.1% 2me | - | - | 200ul |
Total | 200 mL | 200 mL | 200 mL |
- 7/13: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 15 min, and then seeded 1:6 (three wells) onto feeder free system (now become P10).
- 7/14(Day 0): PBS wash twice and then add induction media to each well.
- 7/15-7/16: Take pictures. Cells look ok. Scheduled to change media three days after induction.
- 7/17 (Day 3): Take pictures. Observed some cell death. Change media.
- 7/18: Take pictures. Number of cell death were under control. Important finding: Cell death occurred less in 5% serum group. This might suggests that cell death may be due to lack of nutrition. Managed to change induction media daily hereafter.
- 7/19-7/23: Change media and take pictures daily.