TABLE 4.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6A(v)	No visible cells in the interface	Low amount of starting material	Collect the interface together with Biocoll solution
6A(ix)	Low yield of isolated cells	Low amount of starting material	Increase the number of animals; use older or larger animals
		Insufficient dissociation of kidney marrow	Dissociate the marrow more extensively, and split kidney marrow from multiple animals into several tubes
6B(vi)	Low number of sorted cells	Rare population of gated cells	Change the gating strategy
		Cells do not enter the sorting tube	Recalibrate the FACS instrument
		Cells stick to the wall of the sorting tube, dying during the sorting process	Coat the wall of collection tubes with medium, and re-coat it during the sort if needed
		Cells die during the sorting process	Make sure to keep the cells chilled during the sorting process
12	No or low colony growth	Low number of seeded cells	Increase the number of seeded cells
		Ineffective cytokine stimulation	Increase the effective concentration of cytokine tested or test cooperation between several cytokines
		Unhealthy or inbred fish	Test different fish
		Methylcellulose too dense	Prepare fresh 2% (wt/vol) methylcellulose stock and mix new complete methylcellulose
	Colonies are present even in a negative control	Colony density is too high	Plate fewer cells; when over-plated, cells often generate microenvironments that help them survive and proliferate even without the presence of exogenous supporting factors
		Yeast or bacterial contamination producing microbial colonies	Follow sterile techniques, especially during kidney dissection; use sterile forceps when removing kidney marrow; test for the presence of microbial organisms in a culture medium, especially in carp serum
	Large chunks present in methylcellulose	Methylcellulose stock contains insoluble pieces of methocel	Swirl methylcellulose vigorously after bringing it to a boil during its preparation. Allow the methylcellulose stock to sediment for 1–2 d before using it for mixing the complete methylcellulose. Note: it is common for methylcellulose to contain a small amount of insoluble clumps
	Cells are concentrated on a single area within the well	Inefficient mixing of cell– methylcellulose mixture	Mix the cell–methylcellulose mixture more thoroughly
13	Inability to isolate single colonies	Colony density is too high	Plate fewer cells