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 (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

---

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.
  • The tissue should "stick" together after 20 minutes. Shake every 12-20 minutes to disperse the tissue pieces.
• Put the tube on a rack to let the remaining tissue pieces to settle down at the bottom.
• Transfer the clear 2mL solution on the top to a 30um filter cup on a 15mL falcon tube.
• Mechanically dissociate the remaining tissue by pipetting the remaining 1mL solution with P1000 tip 15-20 times.
• Transfer the solution to the same filter cup.
• To stop the reaction, wash the digestion tube with 3mL HS-AMEM and transfer the medium to the same filter.
  • Collect the filtered solution from the bottom of the filter.
• Spin down the cells, 300x rcf, 20 degree, 5 minutes.
• Remove supernatant and wash with 5mL 0.7xPBS.
• Spin down the cells, 300x rcf, 20 degree, 5 minutes.
• The sample is ready for downstream applications.
  • For FACS
  • scRNA-seq
  • Cell transplantation