Difference between revisions of "LiberaseTM dissociation"

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= Preparing reagents =
 
= Preparing reagents =
 
----
 
----
 +
== 0.7x PBS ==
 +
*Dilute 1x PBS to 0.7x with dH2O
  
<li>'''0.7x Phosphate Buffer Saline (PBS)'''<\li>
+
== Liberase TM solution ==
1x PBS 35 mL
+
*Thaw stock Liberase TM (100x) aliquots on ice.
dH2O 15 mL
+
*30uL Stock Liberase TM
Total 50 mL
+
*2970uL 0.7x PBS in a FACS tube
Store at room temperature. Chill on ice before use.
+
-> chill on ice
  
'''Stock Liberase TM (26 Wünsch Unit (WU)/mL)'''
+
== AMEM(0) ==
Liberase TM 50mg
+
*114.3mL F12:DMEM+Glutmax
dH2O            10mL
+
*1.5mL Sodium pyruvate (100mM)
Store in 50uL aliquots at -80 degree
+
*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.
 +
**[[ELISA]]
 +
**[[For_scRNA-seq|scRNA-seq]]
 +
**[[For_transplantation|Cell transplantation]]

Latest revision as of 21:22, 18 July 2022

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.