LiberaseTM cell dissociation for axolotl/frog limb cells

Prepare Reagents

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0.7x PBS

• Dilute 1x PBS to 0.7x with dH2O

-> Chill on ice before use.

Liberase TM solution

• 40uL Stock Liberase TM (100x, 26WU/mL)
• 3960uL 0.7xPBS in a FACS tube

-> Chill on ice before use.

AMEM(0) (serum-free medium)

• 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

-> Total 150mL, filter.

-> Chill on ice before use.

High-Serum AMEM (HS-AMEM)

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

-> Total 200mL, filter.

-> Chill on ice before use.

Liberase TM cell dissociation on amphibian limb tissue

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• Collect the limb tissue to dissociate into a new 100mm dish.
• Wash the tissue in a 60mm dish with 0.7x PBS to remove blood and debris.
• Transfer the tissue into a new 60mm dish with fresh 0.7x PBS.
• Remove the skin.
• Transfer the limb mesenchyme into a new 60mm dish. Remove as much liquid as possible.
• Chop the tissue into <500mm^3 cubes with a #21 scalpel.
• Transfer the tissue pieces into the Liberase TM solution.
• Rotate on a wheel 40-50 minutes, room temperature. Vigorously shake every 15-20 minutes to disperse the tissue.
  • The tissue will start to clump together after ~20 minutes.
• Put the tube on a rack and wait until the remaining tissue pieces to settle down.
• Collect the cleared, top 3mL solution and filter through a 30mm filter cup on a 15mL falcon tube.
• Mechanically dissociate the remaining tissue by pipetting the remaining 1mL solution 15-20 times with a P1000 tip.
• Filter the remaining solution with the same 30mm filter cup.
• Stop the enzymatic reaction by washing the FACS tube with 4mL HS-AMEM, and then filter the HS-AMEM with the same 30mm filter cup.
• Collect the remaining solution at the bottom of the filter cup into the 15mL falcon tube, gently mix.

-> If doing scRNA-seq directly, continue with these steps.

• Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.
• Wash the cell pellet with 5mL 0.7xPBS
• Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.
  • The sample is ready for downstream applications:

-> for FACS

-> for cell transplantation