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== | =='''Seeding density test (OSG_E1)'''== | ||
*'''Purpose:''' To | *'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation | ||
* | <br> | ||
** | === Test #1 (OSG_E1_T1) === | ||
* | *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). | ||
=='''Medium composition test (OSG_E2)'''== | |||
*'''Purpose:''' To test the effects of osteogenic differentiation media on mESCs | |||
<br> | |||
=== Test #1 (OSG_E2_T1)=== | |||
*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 | ||
|- | |- | ||
| | | b-GP ||2ml of 1M stock||10mM | ||
|- | |- | ||
| | | PenStrep ||2ml||1% | ||
|- | |- | ||
| style="background:#f0f0f0;"| | | style="background:#f0f0f0;"|Total | ||
| style="background:#f0f0f0;"| | | style="background:#f0f0f0;"|200 mL | ||
| style="background:#f0f0f0;"| | | 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). | |||
=== Test #2 (OSG_E2_T2) === | |||
*Date: 7/13/2012 ~ 7/27/2012 | |||
*Cells: P10 InsCAGGsAlov3 (mES brainbow cells) | |||
*Osteogenic differentiation media: | |||
( | Basal media were changed from MEM to DMEM. Different concentration of serum were tested. | ||
{| {{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) | |- | ||
| 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. | |||
*7/19-7/26: Change media and take pictures daily. | |||
*7/27: Perform Alizarin Red S staining. (Fixed with 3.7% paraformaldehyde for 20 min; washed with Ca- Mg- PBS; stained for 1 hour.) | |||
( | === Test #3 (OSG_E2_T3) === | ||
*Date: 7/30/2012 ~ 8/3/2012 | |||
*Cells: P12 InsCAGGsAlov3 (mES brainbow cells) | |||
*Osteogenic differentiation media: 0, 2.5, 5, 7.5, 10, 12.5, and 15% serum in osteogenic differentiation media were tested. | |||
*7/30: P11 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:6 onto gelatin coated 6-well plates (now become P12). | |||
*7/31(Day 0): PBS wash twice and then add induction media to each well. | |||
*Change media daily. | |||
*8/3 (Day 3): Cell proliferated too fast. Cell colony became very big. It's really difficult to observe osteogenic differentiation. Therefore, cells were discarded. | |||
*Cell seeding density needs to be adjusted. Try 1:12 or 1:18 next time. | |||
** | === Test #4 (OSG_E2_T4) === | ||
*Date: 8/3/2012 ~ ongoing work | |||
*Cells: P13 InsCAGGsAlov3 (mES brainbow cells) | |||
*Osteogenic differentiation media: 5, 7.5, 10, 12.5, and 15% serum in osteogenic differentiation media were tested. | |||
*8/3: P12 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:8 onto gelatin coated 6-well plates (now become P13). | |||
*8/4 (Day 0): PBS wash twice and then add induction media to each well. 0%, 1%, 5%, 10%, 15% OSG: each for five 6-wells. | |||
*8/5 (Day 1): PBS wash twice and change media. There are cell death occurred in 0% and 1% serum. So, made some adjustment: 0% were changed into 7.5% OSG; 1% were changed into 12.5% OSG. | |||
*Change media daily (PBS wash once). | |||
*''' | =='''Induction efficiency test (OSG_E3)'''== | ||
*'''Purpose:''' To test the osteogenic differentiation efficiency of mESCs-derived osteoblasts | |||
<br> | |||
=== Test #1 (OSG_E3_T1)=== | |||
*Date: 7/30/2012 ~ 8/4/2012 | |||
( | *Cells: P12 InsCAGGsAlov3 (mES brainbow cells) | ||
*Osteogenic differentiation media: 5% serum in osteogenic differentiation media. | |||
*7/30: P11 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:6 onto gelatin coated 25Ts (now become P12). | |||
7/31(Day 0): PBS wash twice and then add induction media to each well. | |||
Change media daily. | |||
8/4 (Day 4): Like OSG_E2_T3, also observed lots of cell death. Cell proliferated too fast. Cell colony became very big. Therefore, cells were discarded. Cell seeding density needs to be adjusted. Try 1:12 or 1:18 next time. | |||
( | === Test #2 (OSG_E3_T2)=== | ||
*Date: 8/3/2012 ~ ongoing work | |||
*Cells: P13 InsCAGGsAlov3 (mES brainbow cells) | |||
*Osteogenic differentiation media: 5% serum in osteogenic differentiation media. | |||
*8/3: P12 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:18 onto gelatin coated 25T (now become P13). | |||
*8/4 (Day 0): PBS wash twice and then add induction media. | |||
*Change media daily (PBS wash once). | |||
*8/7 (Day 3): Collect sample from one 25T. Store at -80C. | |||
** | === Test #3 (OSG_E3_T3)=== | ||
*Date: 8/6/2012 ~ ongoing work | |||
*Cells: P14 InsCAGGsAlov3 (mES brainbow cells) | |||
*Osteogenic differentiation media: 5% serum in osteogenic differentiation media. | |||
*8/6: P13 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 30 min, and then seeded 1:18 onto gelatin coated 35mm dishes (now become P14). | |||
*8/7 (Day 0): PBS wash twice and then add induction media (5% serum). Perform Alizarin red S staining in one 35mm dish. (Fix for 30 min; Stain for 20 min.) | |||
*Change media daily (PBS wash once). |
Latest revision as of 01:19, 9 August 2012
Seeding density test (OSG_E1)[edit]
- Purpose: To determine the seeding density of mESCs for osteogenic differentiation
Test #1 (OSG_E1_T1)[edit]
- 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).
Medium composition test (OSG_E2)[edit]
- Purpose: To test the effects of osteogenic differentiation media on mESCs
Test #1 (OSG_E2_T1)[edit]
- 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 (OSG_E2_T2)[edit]
- Date: 7/13/2012 ~ 7/27/2012
- Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media:
Basal media were changed from MEM to 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.
- 7/19-7/26: Change media and take pictures daily.
- 7/27: Perform Alizarin Red S staining. (Fixed with 3.7% paraformaldehyde for 20 min; washed with Ca- Mg- PBS; stained for 1 hour.)
Test #3 (OSG_E2_T3)[edit]
- Date: 7/30/2012 ~ 8/3/2012
- Cells: P12 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media: 0, 2.5, 5, 7.5, 10, 12.5, and 15% serum in osteogenic differentiation media were tested.
- 7/30: P11 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:6 onto gelatin coated 6-well plates (now become P12).
- 7/31(Day 0): PBS wash twice and then add induction media to each well.
- Change media daily.
- 8/3 (Day 3): Cell proliferated too fast. Cell colony became very big. It's really difficult to observe osteogenic differentiation. Therefore, cells were discarded.
- Cell seeding density needs to be adjusted. Try 1:12 or 1:18 next time.
Test #4 (OSG_E2_T4)[edit]
- Date: 8/3/2012 ~ ongoing work
- Cells: P13 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media: 5, 7.5, 10, 12.5, and 15% serum in osteogenic differentiation media were tested.
- 8/3: P12 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:8 onto gelatin coated 6-well plates (now become P13).
- 8/4 (Day 0): PBS wash twice and then add induction media to each well. 0%, 1%, 5%, 10%, 15% OSG: each for five 6-wells.
- 8/5 (Day 1): PBS wash twice and change media. There are cell death occurred in 0% and 1% serum. So, made some adjustment: 0% were changed into 7.5% OSG; 1% were changed into 12.5% OSG.
- Change media daily (PBS wash once).
Induction efficiency test (OSG_E3)[edit]
- Purpose: To test the osteogenic differentiation efficiency of mESCs-derived osteoblasts
Test #1 (OSG_E3_T1)[edit]
- Date: 7/30/2012 ~ 8/4/2012
- Cells: P12 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media: 5% serum in osteogenic differentiation media.
- 7/30: P11 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:6 onto gelatin coated 25Ts (now become P12).
7/31(Day 0): PBS wash twice and then add induction media to each well. Change media daily. 8/4 (Day 4): Like OSG_E2_T3, also observed lots of cell death. Cell proliferated too fast. Cell colony became very big. Therefore, cells were discarded. Cell seeding density needs to be adjusted. Try 1:12 or 1:18 next time.
Test #2 (OSG_E3_T2)[edit]
- Date: 8/3/2012 ~ ongoing work
- Cells: P13 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media: 5% serum in osteogenic differentiation media.
- 8/3: P12 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:18 onto gelatin coated 25T (now become P13).
- 8/4 (Day 0): PBS wash twice and then add induction media.
- Change media daily (PBS wash once).
- 8/7 (Day 3): Collect sample from one 25T. Store at -80C.
Test #3 (OSG_E3_T3)[edit]
- Date: 8/6/2012 ~ ongoing work
- Cells: P14 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media: 5% serum in osteogenic differentiation media.
- 8/6: P13 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 30 min, and then seeded 1:18 onto gelatin coated 35mm dishes (now become P14).
- 8/7 (Day 0): PBS wash twice and then add induction media (5% serum). Perform Alizarin red S staining in one 35mm dish. (Fix for 30 min; Stain for 20 min.)
- Change media daily (PBS wash once).