Table V.

Table of troubleshooting tips for papain dissociation protocol.

Issue	Possible cause(s)	Advice for solution
Poor tissue dissociation	Enzymatic failure	Ensure the papain enzyme is pre-warmed to 37°C prior to use, and make sure the enzyme is in date. Use freshly prepared papain in HBSS for each dissociation.
	Tissue rigidity	Increase the frequency of the manual dissociation and pipette up and down with 5-mL glass serological pipettes that have been pre-coated with HBSS solution. For older ovaries, the tissue should be minced with a scalpel blade or mincing scissors prior to dissociation. Perform mincing in a glass dish and transfer the minced ovary using a 5-ml glass serological pipette.
Loss of cell viability during the dissociation	Excessive tissue dissociation	Decrease the frequency of the manual dissociation and pipette up and down with 5-ml glass serological pipettes that have been pre-coated with HBSS solution. Decrease the concentration of papain used by 5% to ensure cell viability is maintained. Perform all dissociation steps following the enzymatic dissociation at room temperature, and once completed, immediately snap freeze or fix cells for subsequent procedures.
Cell clumping/a failure to get a single cell suspension	Insufficient tissue dissociation	Increase the frequency of the manual dissociation and pipette up and down with 5-ml glass serological pipettes that have been pre-coated with HBSS solution. Increase the percentage of BSA to 0.05% to prevent cell clumping.
Small/no pellet after dissociation	Cell loss	Ensure pipette tips and serological pipettes are all coated with Hank’s solution before coming into contact with cells. Gently remove the supernatant from the cell pellets after the centrifugations, rather than tipping the tube. This is most effectively done by pipetting off the liquid. When possible, pellet the cells in 15-mL tubes and avoid using round-bottom tubes in the centrifugation steps.