LiberaseTM dissociation
Preparing reagents
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 (56C, 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
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