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
iPS media
500 ml
- 390 ml DMEM/F12
- 100 ml KOSR
- 5 ml PSG
- 5 ml NEAA, 100x
- 500 ul b-mercaptoethanol
filter
N2 Neural Induction media
500 ml
- 490 ml DMEM/F12
- 5 ml NEAA, 100x
- 5 ml N2 supplement (at -20oC)
- 200 ul 5 mg/ml heparin (at 4oC)
filter
N2/B27 Neural Induction media
500 ml
- 480 ml DMEM/F12
- 5 ml NEAA, 100x
- 5 ml N2 supplement (at -20oC)
- 10 ml B27 supplement (at -20oC)
- 200 ul 5 mg/ml heparin (at 4oC)
filter
Neural Differentiation media
500 ml
- 480 ml Neurobasal
- 5 ml NEAA, 100x
- 5 ml N2 supplement (at -20oC)
- 10 ml B27 supplement (at -20oC)
filter
NPC media
500 ml
- 350 ml DMEM
- 150 ml F12
- 5 ml sodium pyruvate (if DMEM says “ – Na pyruvate”)
- 5 ml PSG
- 10 ml B27 supplement (at -20oC)
filter
MEF media
500 ml
- 440 ml DMEM
- 5 ml P/S
- 5 ml L-glutamine
- 50 ml FBS
filter