LiberaseTM dissociation

Preparing reagents

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0.7x PBS

• Dilute 1x PBS to 0.7x with dH2O

Liberase TM solution

• Thaw stock Liberase TM (100x) aliquots on ice.
• 30uL Stock Liberase TM
• 2970uL 0.7x PBS in a FACS tube

-> chill on ice

AMEM(0)

• 114.3mL F12:DMEM+Glutmax
• 1.5mL Sodium pyruvate (100mM)
• 1.5mL B27 supplement
• 1.5mL Insulin (1 mg/mL)
• 1.5mL MEM Non-Essential Amino Acid (NEAA)
• 1.5mL Penicillin/Streptomycin (10000 U/mL)
• 28.2mL Sterile ddH2O

-> 150mL total, filter. -> Chill on ice

High-serum AMEM (HS-AMEM)

• 125mL Minimum essential medium (MEM)
• 20mL Heat-inactivated fetal bovine serum (56C, 30 min)
• 2mL Insulin (1 mg/mL)
• 2mL Glutamine (200mM)
• 2mL Penicillin/Streptomycin (10000 U/mL)
• 49mL Sterile ddH2O

-> 200mL total, filter. -> Chill on ice

Liberase TM dissociation

Dissociation of Frog Limb Bud Cells Timing: 2-3 hours

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This step describes the Liberase-based cell dissociation method to obtain high quality cell suspension for downstream applications. We recommend performing the following steps in a sterile environment.

• Anaesthetise the animals and collect the limb bud tissue of your desired stages in a new 100mm dish.
• Transfer the limb bud tissue into a new 60mm dish with fresh 0.7x PBS
• Remove the skin using a pair of autoclaved fine tweezers.
• Transfer the limb bud mesenchyme into a new 60mm dish and remove as much liquid as possible
• Chop the limb bud mesenchyme into <500um^3 cubes
• Transfer the limb bud pieces into Liberase TM solution
• 40-50 minutes on the wheel, room temperature
  • Shake every 12-20 minutes