Difference between revisions of "LiberaseTM cell dissociation for axolotl/frog limb cells"
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(Created page with "= Prepare Reagents = ---- ‘’’0.7x PBS’’’ *Dilute 1x PBS to 0.7x with dH2O :-> Chill on ice before use") |
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= Prepare Reagents = | = Prepare Reagents = | ||
---- | ---- | ||
| − | + | === 0.7x PBS === | |
*Dilute 1x PBS to 0.7x with dH2O | *Dilute 1x PBS to 0.7x with dH2O | ||
| − | :-> Chill on ice before use | + | :-> 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 = | ||
| + | ---- | ||
| + | *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 [[for scRNA-seq|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]] | ||
Latest revision as of 22:27, 18 July 2022
Prepare Reagents
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
- 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: