LiberaseTM dissociation

Preparing reagents

Dissociation of Frog Blastema 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.

1. Thaw stock Liberase TM aliquots on ice. 2. Using benzocaine to anesthetize the eight CAGGs-Venus animals with their legs amputated 14 days prior in compliance with the local ethic guidelines. 3. Collect the blastema plus stump tissue in a new 100mm dish: a. Isolate the whole upper leg from the most proximal region and collect the cut leg in a new 60mm dish with fresh 0.7x PBS (Figure 2A). b. Remove the skin using a pair of autoclaved fine tweezers (Figures 2B and 2C). Depending on the downstream applications, the skin layer could also be processed in parallel with this protocol. c. Rinse the leg with 0.7x PBS and transfer the sample to a new 60mm dish on ice with fresh 0.7x PBS. 4. Repeat Step 3 until the blastemas are collected from all donor frogs. 5. Collect blastema tissues: a. Remove the muscle bundles from each leg carefully with spring scissors under a fluorescence stereoscope (Figures 2D-2F). CAGGs promoters have a stronger activity in muscle tissues than in the blastema or bone tissues. b. Isolate the blastema tissue using a scalpel. c. Rinse and transfer the blastema to a new 60mm dish on ice with fresh 0.7x PBS. 6. Repeat Step 5 until all the blastema tissues are collected (Figure 2G). 7. Prepare 4mL Liberase solution and keep on ice. (See Materials and equipment) 8. Chop the blastemas into fine pieces: a. Transfer the blastema tissue into the lid of a 60mm dish. b. Remove the residual 0.7x PBS as much as possible using a pipette. c. Chop the tissue into <1mm cubes using a size 21 scalpel. Avoid over-chopping (Figure 2H). d. Gather the tissue cubes into a pile using the scalpel and transfer the tissue cubes into the Liberase solution using forceps. 9. Digest the tissue on a rotating wheel for 45-50 minutes at room temperature.

Note: We have compared multiple commonly available enzymes to digest limb tissues. Liberase TM has out-performed Liberase TL, Liberase DL, Collagenase I, Dispase, and Trypsin, in terms of cell yield and survival rate.

CRITICAL: Chopping tissues into small pieces before enzymatic digestion is critical. Large tissue pieces will not be fully digested in this time frame, resulting in a poor cell yield. Longer digesting times (>1 hour) are also not recommended as it increases cell death.

CRITICAL: The volume of the tissue should be no more than 15% of the total volume of Liberase solution (Figure 2I). Higher tissue-to-solution ratio leads to a poor yield.

CRITICAL: The tissue pieces will start clumping together after around 10-15 minutes (Figures 2J and 2K). Remove tubes from the rotator and flip the tube to invert the liquid to get better dispersal every 10-15 minutes. The tissue pieces should be significantly smaller and transparent at the end of enzymatic reaction.

10. Mechanical dissociation of cells and filtering a. Pre-wet a new 30m MACS® SmartStrainer with 0.7x PBS. b. Put the strainer on a new 15mL Falcon tube on ice. c. After enzymatic digestion, place the tube of digested tissue upright on ice, letting the remaining tissue pieces fall to the bottom of the tube by gravity. d. Transfer 2mL of the supernatant to the strainer to allow the liquid filtered into the 15mL Falcon tube. e. Pipette the remaining solution up and down 10-20 times using a P1000 pipette tip to mechanically disintegrate the remaining tissue pieces. f. Filter the solution into the same 15mL Falcon tube as in Step 10-d. g. Rinse the reaction tube with 2mL HS-AMEM to collect the remaining cells. h. Filter the solution into the 15mL Falcon tube. i. Rinse the strainer with 2mL fresh HS-AMEM. j. Collect the solution (200-400L) stuck under the filter and add to the filtered cell suspension in the 15mL Falcon tube before trashing the filter (Figure 2L). k. Gently mix the filtered solution by pipetting and keep it on ice. 11. Repeat Step 10 if there are multiple reactions.

Optional: For single cell RNA-sequencing applications, filter the cell suspension again using a 10m filcon to remove multiplets. Omit this step when working with axolotl cells due to the large cell size.

CRITICAL: Refrain from pipetting the solution more than 30 times even if the tissue pieces remain visible. Excessive pipetting may lead to low viability.

12. Spin down the cells using a swing-bucket centrifuge at 300x rcf for 5 minutes at 12℃. 13. Remove the supernatant and resuspend the pellet with 2mL of 0.7x PBS. 14. Spin down the cells using a swing-bucket centrifuge at 300x rcf for 5 minutes at 12℃.

Optional: For single cell RNA-sequencing applications, repeat Steps 12 to 14 at least once (2 times in total).

15. Remove supernatant and resuspend the cell pellet in 200L of AMEM(0) and keep it on ice. 16. Count the cells and determine cell viability using trypan blue: a. Mix 5L cell suspension with 5L trypan blue. b. Load the cell/trypan blue solution into a hemocytometer and calculate the following two parameters following the manufacturer’s instructions:  Cell concentration.  Cell death rate.

Note: Adjusting the volume base on the pellet size to achieve a cell concentration between 300-1000 cells/L. This ensures enough cells for 6-18 injections in one sealed pipette tip, as the maximum loading volume in a sealed pipette is 200L.

CRITICAL: 20,000-30,000 cells per blastema and >90% viable cells are expected. Consider repeating the procedure with modifications if the total cell number is too low or the survival rate is <80%. (See Troubleshooting)