LiberaseTM dissociation
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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.
- 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.