Table III.

Table of troubleshooting tips for collagenase dissociation protocol.

Issue	Possible cause(s)	Advice for solution
Ovary tissue not breaking down during enzymatic dissociation step	Enzymatic failure	Ensure reagents stored and used correctly, that solution is completely combined and that the total quantity of tissue is ≤20 mg
	Tissue rigidity	Older ovaries are more fibrous and may require more forceful mechanical agitation when pipetting puncture/tearing with a needle while in the dissociation solution. Additionally, an extra 15-min incubation in dissociation solution for a total 75 min may suffice
Cells adhering to plate after trypsinisation and wash	Insufficient digestion	Check cells before and after 5-minute incubation with trypsin to ensure they have lifted from the plate. Incubate for a further minute if required. Additionally, tapping plate firmly against a solid surface and pipetting directly onto the plate with force may dislodge any rounded cells remaining on the bottom of the plate
Loss of cell viability after overnight culture	Insufficient nutrients	Ensure ovary culture media is kept at 4°C when not in use, and always heated to 37°C before use. Doubling the proportion of FCS in media to 10% may also increase viability
	Overcrowding on plate	Dilute cell suspension before plating and seed multiple plates, or switch seeding cells in a flask (T25 or T75)
Small/no pellet after collection	Cell loss	Ensure pipette tips and serological pipettes are all coated with Hank’s solution before coming into contact with cells. When not in use, store Hank’s solution at 4°C, equilibrate to room temp before use, and do not use following storage exceeding 2 weeks