Tina:Osteogenic differentiation on mES brainbow cells (OSG): Difference between revisions

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==Lentiviral production==
==Seeding density test==
*'''Purpose:''' To generate and concentrate Cre lentivirus
*'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation
*'''Components:'''  
*'''Test #1:'''
**Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control)
**Date: 7/5/2012 ~ 7/7/2012
**Packaging plasmids: psPAX2 and pMD2.G
**Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
*'''Batch #1:'''
**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: 6/26/2012 ~ 6/29/2012
**7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.
**Cells: 293T 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).
**Virus packaging components:  
 
 
==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;"|'''Cre lentivirus'''
| align="center" style="background:#f0f0f0;"|'''Volumn'''
| align="center" style="background:#f0f0f0;"|'''Control'''
| align="center" style="background:#f0f0f0;"|'''Final concentration'''
|-
|-
| pLenti CAG Cre IRES Puro ||90ug||-
| MEM ||193ml||
|-
|-
| pLenti CMV GFP Puro ||-||30ug
| Serum ||2ml||1%
|-
|-
| psPAX2 (packaging plasmid) ||60ug||20ug
| Dexamethasone ||200ul of 0.1mM stock||0.1uM
|-
|-
| pMD2.G (packaging plasmid) ||30ug||10ug
| Ascorbic acid ||1ml of 10mg/ml stock ||50ug/ml
|-
|-
| style="background:#f0f0f0;"|Transfected 293T cell number
| b-GP ||2ml of 1M stock||10mM
| style="background:#f0f0f0;"|27*10^6 cells
|-
| style="background:#f0f0f0;"|9*10^6 cells
| PenStrep ||2ml||1%
|-
| style="background:#f0f0f0;"|Total
| style="background:#f0f0f0;"|200 mL
| style="background:#f0f0f0;"|
|}
|}
**Virus production: Exp was carried out at Dr. Yang Xu’s Lab (with Dr. Blue Lake’s help). 293T cells were seeded on 6/26 and transfected on 6/27. Virus were collected and concentrated on 6/29. Aliquot (105 ul) virus are stored at -80゚C at CERC.
**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).  
==LentiGFP infection==
**Add induction media to each well on 7/4 to observe the cell morphology changes at the beginning of osteogenic differentiation.
*'''Purpose:''' To confirm the efficiency of virus packaging
**7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.)
*'''Test #1:'''
**7/6: Possible cause of cell death: (1)chemical concentration is too high (toxic). (2)serum concentration is too low.
**Date: 7/2/2012 ~ 7/6/2012
Further tests should be arranged (with lower chemical concentration and/or higher serum concentration).
**Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto one gelatin coated glass-well.
**'''Components:''' Lentiviruses: pLenti CMV GFP Puro
**Infection on 7/3:
(1)Take pictures of cells before viral infection
 
(2)Change media of each well (2 ml/well)
 
(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency
 
(4)Add 50 ul LentiCre virus in culture medium, and then mix gently
 
(5)Put cells back into incubator
 
(6)Take pictures of cells 2-3 days after infection.
 
**Results:
Observation: Successful infection of GFP vectors. Both mESCs and MEFs are expressing GFPs now. Efficiency is higher in MEFs.
Pictures:
 
 
 
==LentiCre infection==
*'''Purpose:''' To confirm the effect of Cre recomninase in mES brainbow cells
*'''Test #1:'''
**Date: 7/1/2012 ~ 7/5/2012
**Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells).
**'''Components:''' Lentiviruses: pLenti CAG Cre IRES Puro
**Infection on 7/3:
(1)Take pictures of cells before viral infection
 
(2)Change media of each well (2 ml/well)
 
(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency
 
(4)Add 0/10/25/50 ul LentiCre virus in culture medium, and then mix gently
 
(5)Put cells back into incubator
 
(6)Take pictures of cells 2-3 days after infection.


**Results:




*'''Test #2:'''
*'''Test #2:'''
**Date: 7/2/2012 ~ 7/6/2012
**Date: 7/13/2012 ~ ongoing work
**Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto gelatin coated glass-well (total: four wells).
**Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
**'''Components:''' Lentiviruses: pLenti CAG Cre IRES Puro
**Osteogenic differentiation media:
**Infection on 7/3:
Basal media were changed from MEM into DMEM. Different concentration of serum were tested.
(1)Take pictures of cells before viral infection
{| {{table}} border=1
 
| align="center" style="background:#f0f0f0;"|''Component (volumn)/group''''
(2)Change media of each well (2 ml/well)
| align="center" style="background:#f0f0f0;"|''5% serum''''
 
| align="center" style="background:#f0f0f0;"|''15% serum''''
(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency
| align="center" style="background:#f0f0f0;"|''2me''''
 
|-
(4)Add 0/10 ul LentiCre virus in culture medium, and then mix gently
| DMEM ||193ml||193ml||193ml
 
|-
(5)Put cells back into incubator
| Serum ||5%(10ml)||15%(30ml)||15%(30ml)
 
|-
(6)Take pictures of cells 2-3 days after infection.
| 0.1 uM Dexamethasone ||200ul of 0.1mM stock||200ul of 0.1mM stock||200ul of 0.1mM stock
 
|-
**Results:
| 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.