Table 20.1.3.

Troubleshooting Guide for Use of Mouse Model for Human Melanoma

Problem	Possible cause	Solution
Poor tumor take	Mycoplasma infection of tumor cultures	Eliminate mycoplasma with ciprofloxacin or thaw vial with fresh tumor sample
	Overtrypsinization of tumor cells	Dilute out trypsin with large volume of HBSS as soon as tumor cells dislodge; pellet and discard supernatant
	Tumor cells not kept on ice	Keep tumor on ice at all time
	Injected too few tumor cells	Increase inoculum of tumor cells
Poor treatment upon adoptive transfer of CTL	Low potency or specificity of CTL	Test CTL in vitro directly prior to transfer to ensure specificity
	Too few CTL transferred	Transfer more CTL, or administer IL-2
Poor CTL growth	Too little IL-2 in cultures	Prepare new IL-2 stock, check type of units
	Poor FBS quality	Test different FBS lots
	“Dirty” cultures	Purify on Ficoll to eliminate debris
	Over- or understimulation	Restimulate every 7-10 days
	Overcrowding of culture wells	Split once CTL exceed 50% confluency
Poor CTL specificity/high background (often irreversible)	Too much IL-2 in cultures	Keep [IL-2] between 30-60 IU/ml
	“Dirty cultures”	Ficoll to eliminate debris
	Progressive deterioration of specificity, large, granular, fast-growing cells	Discard culture and thaw new vial or establish new culture from immunized mice
No induction of vitiligo and/or antibodies upon two immunizations with rVVmTRP-1	Poor viral titer	Retiter viral stock
	Mixup of rVV stocks	Check expression by immunostaining of infected cells