Mouse iPSC general culture protocol

PMEF media
  • 450mL DMEM
  • 50mL FBS
  • 5mL L-glut (200mM)
  • 5mL Pen/Strep (100X)

Sterile filter into 500mL bottle and store at 4C. Good for 6 months. Keep a 50mL tube for easier warming.

ES culture media (ESCM)
  • 780mL DMEM
  • 200mL ES-qualified FBS
  • 10mL NEAA (100X)
  • 10mL Nucleosides (100X)
  • 1mL Pen/Strep (1000X)

Prepare and sterile filter into 1L bottle. Make 50mL aliquots. Good for 6 months.

ESCM/2i
  • 50mL ESCM
  • 500uL L-glut
  • 50uL CHIR99021 inhibitor (3mM, aliquots in –20C box)
  • 50uL PD0325901 (1mM, aliquots in –20C box)
  • 50uL mLIF (1e6 Units/mL)
  • 50uL B-ME (1000X working stock)

Prepare and carefully invert to mix. Stable for 2wks at 4C.

iPSC freezing media

50% FBS + 40% base media (ESCM, SFES, or PMEF) + 10% DMSO

Prepare as needed. Can store at 4C up to a month.

STOP buffer

50% FBS + 50% IMDM

Thaw an aliquot of regular FBS (25mL) and add 25mL IMDM. Invert to mix. Store at 4C.

mPSC reprogramming media
  • 395mL DMEM/F-12
  • 100mL KnockOut Serum Replacement
  • 5mL NEAA (100X)
  • 500uL B-ME working stock (1000X, final concentration 55uM)
  • 500uL mLIF (1e6 Units/mL)

Sterile filter into 500mL bottle and store at 4C. Good for up to 3wks. Can keep a 50mL tube for easier warming.

SFES base media
  • 500mL DMEM/F-12
  • 500mL Neurobasal media
  • 6.7mL BSA (7.5%)
  • 1mL Pen/Strep (1000X)
  • 10mL B27 supplement (1 vial, stored –20C)
  • 5mL N2 supplement (1 vial, stored –20C)

Sterile filter into 1L bottle. Make 50mL aliquots and store protected from light. Good at 4C for 6 months.

2i media
  • 50mL SFES base media
  • 500uL L-glut (200mM, aliquots in ES inhibitors box at –20C)
  • 50uL CHIR99021 inhibitor (3mM, aliquots in –20C box)
  • 50uL PD0325901 (1mM, aliquots in –20C box)
  • 50uL mLIF (1e6 Units/mL)
  • 0.63uL Monothioglycerol (MTG, kept in TC hood)

Prepare and carefully invert to mix. Stable for 2wks at 4C.

Media + hygro

Add 2.8uL hygro working stock (50mg/mL) for every 1mL media. Final concentration is 140ug/mL. Store at 4C (can keep for as long as you would the base media).