iPSC Recipes

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