iPSCs
Mef Media (500 mls)
DMEM 440 mls
PSG 10 mls
FBS 50 mls
*sterile filter
Embryoid Body Protocol
iPS cell media (500 mls)
DMEM/F-12 390 mls
KOSR 100 mls
MEM NEAA 5 mls
B- mercaptoethanol 3.5 ul
L-glutamine 2.5 mls
*sterile filter
N2 Neural Induction Medium (500 mls)
DMEM/F12 490 mls
N2 5 mls
MEM NEAA 5 mls
Heparin (4C) 500 ul
* sterile filter
N2/B27 Neural Induction Medium (post D17 and Rosette Selection) (500 mls)
DMEM/F12 480 mls
N2 5 mls
B27 10 mls
MEM NEAA 5 mls
Heparin 500 ul
*sterile filter
Neuronal Diff (500 mls)
NBM 480 mls
N2 5 mls
B27 10 mls
MEM NEAA 5 mls
*sterile filter
Dispace (10 mls)
Dispace 10 mg
DMEM/F12 10 mls
Matrigel
To aliquot:
- Thaw overnight in ice bucket in 4oC fridge
- Use tips, pipettes, and tubes that are ice-cold
- Aliquot in 1 ml, 300 ul, and/or 100 ul volumes
- Label volume and matrigel lot number on side of tube, “M” on cap
- Usually 7-8 x 1 ml, 7-10 x 300 ul, 0-10 x 100 ul (10 ml total)
- Store aliquots at -20oC
To coat plates:
- Take matrigel out of -20oC just before use
- If matrigel is heated to 37oC, it gels and cannot be resuspended
- Add 20x aliquot volume of sterile-filtered DMEM (e.g. add 20 ml sterile DMEM to 1 ml matrigel aliquot)
- Resuspend by pipetting, ideally with cold pipet
- Apply resuspended matrigel to cell strainer, 40-100 uM
- Use filtered matrigel to coat plates
- 1 ml for 6 well plates, 50 ul for 96 well plates, etc.
FGF-2 diluent
50 ml
0.25g BSA
500 ul 0.1 M DTT
5 ml glycerol
44.5 ml PBS
- Filter, aliquot 5 ml/tube, freeze at -20oC
FGF-2
- Centrifuge FGF tubes prior to opening, 5’ at max speed, 4oC
- Dilute 50 ug FGF-2 in 5 ml FGF-2 diluent (-20oC), pipet up & down to resuspend
- Aliquot 500 ul/cryotube, keep at -20oC
- 1000x for iPSCs, 500x for NPCs
BDNF/GDNF:
- Centrifuge BDNF/GDNF tubes prior to opening, 5’ at max speed, 4oC
- Add 1 ml 0.1% BSA in PBS (filtered, 4oC) to 10 ug, pipet up & down to resuspend
- Aliquot 50 ul/cryotube, keep at -20oC
- 1000x for neural differentiation
IGF-1
- Centrifuge IGF-1 tube prior to opening, 5’ at max speed, 4oC
- Add 1 ml sterile water to 100 ug, pipet up & down to resuspend
- Add 9 ml 0.1% BSA in PBS (filtered, 4oC)
- Aliquot 1 ml/cryotube, keep at -20oC
- 1000x for neural differentiation
cAMP
- Make 1 uM in sterile water
- 4.914 mg cAMP in 10 ml sterile milliQ water, pipet up & down to resuspend
- If you make > 10 ml, keep 10 ml aliquots at -20oC and thaw/re-aliquot these later, 1 ml/tube
- Aliquot 1 ml/cryotube, keep at -20oC
- 1000x for neural differentiation
Heparin
- Make 5 mg/ml in sterile water
- 1000x for NPCs, use 200 ul for N2 or N2/B27 Neural Induction media
ROCK-Inhibitor
- Make 10 mM in DMSO
- Add 1.478 ml iPS room DMSO to 5 mg ROCK-Inhibitor vial, pipet up & down to resuspend
- Aliquot 50 ul/cryotube, keep at -20oC
- Light-sensitive!
- 1000x
ES/iPS Cells
mTeSR media (500 mls)
mTeSR bottle 400 mls
mTeSR supplement 100 mls
P/S 5 mls
*sterile filter
IN Protocol
|
Inhibitor |
Vendor |
Cat no. |
[Stock] |
[Final] |
Dilution |
|
LDN-193189 |
Stemgent |
04-0074 |
500 uM |
100 nM |
1:5000 DMSO |
|
SB431542 |
Tocris |
1614 |
10 mM |
10 uM |
1:1000 ETOH |
|
XAV939 |
Stemgent |
04-00046 |
10 mM |
2 uM |
1:5000 DMSO |
Human KSR Media (500 mls)
DMEM KO 415 mls
KOS 75 mls
MEM NEAA 5 mls
b-mercaptoethanol 500 uls
Glutamax 5 mls
*sterile filter
N2 supplement B Media N2B (500 mls)
DMEM/F12 500 mls
Glutamax 5 mls
20% Dextrose 7.5 mls
N2 Supplement B 5 mls (add after filtration step)
*Sterile filter
*1:100 B27 should be added just prior to use
Neurobasal Media NBM (500 mls)
Neurobasal Medium (NBM) 485 mls
Glutamax 5 mls
20% Dextrose 7.5 mls
MEM NEAA 2.5 mls
*Sterile filter
*1:50 B27 should be added just prior to use
Cortical Neuron Preparation
HBSS- dissection media
1x HBSS with Ca2+ and Mg2+ (Diluted from 10x, filter sterilize)
1xHBSS without Ca2+ and Mg2+(Diluted from 10x, filter sterilize)
DMEM + 5% FBS- plating media
DMEM 475 mls
P/S 10 mls
FBS 25 mls
** Filter sterilize
Neurobasal media- growth media
Neurobasal 500 mls
B27 w/antioxidants 1 vial
Glutamax stock 5 mls
Gentamicin 500 ul
** Filter sterilize