Table 2.

Troubleshooting^a

Problem	Possible cause	Solution
No cross-presentation	No PRR stimulation	Include a PRR agonist, e.g., LPS
	Spontaneous maturation of DCs	Take extra care while handling
		DCs and minimize vibrations
	Antigen (particularly if this is cell-associated antigen from apoptotic cells) was not delivered to the DC	See Blander and Medzhitov, 2006 for a published protocol to verify phagocytosis of apoptotic cells by DCs
Cross-presentation in control/non-stimulated DCs	Non-endotoxin-free reagents used and/or contamination	Test all reagents for endotoxins prior to use
Apoptotic B cell blasts not staining for annexin V	Did not use the proper buffer	Ensure all staining and acquisition is done in the annexin V staining buffer included in the kit
Low yields of DCs	Incubation with RBC lysis buffer is too long	Be very careful to incubate for exactly 1 min with the RBC lysis buffer
Cross-presentation is observed even in the absence of TLR ligands, for instance, when using the microbeads coated with model antigen	Concentration of model antigen is too high. In the presence of high amounts of model antigen, the need for TLR ligands can be bypassed. See Blander and Medzhitov, 2006 where titration with and without LPS was conducted	Carefully titrate the amount of model antigen given to the DCs by creating serial dilutions of the antigen, in whatever form it is being delivered, and evaluate cross-presentation
CD8+ T cells dying after CFSE labeling	CFSE at high concentrations can be toxic to the CD8+ T cells	Use less of the CFSE dye to label CD8+ T cells
CD8+ T cells dying after co-culturing with PFA fixed DCs	Residual PFA has not been adequately washed away from fixed DCs. PFA is highly toxic to living cells, and as such, any residual PFA would kill the CD8+ T cells	Follow protocol to adequately wash away any residual PFA from the fixed DCs. If necessary, include extra washes and longer incubations before co-culture
No cross-presentation occurring in DCs stimulated with E. coli expressing the model antigen of interest	IPTG used to induce expression of the model antigen of interest is old	Use freshly prepared or freshly thawed IPTG to induce expression of the model antigen of interest

^aAbbreviations: DC, dendritic cell; LPS, lipopolysaccharides; PRR, pattern recognition receptor; OVA, ovalbumin; RBC, red blood cell; TLR, Toll-like receptor; CFSE, carboxyfluorescein diacetate sccinimidyl ester; PFA, paraformaldehyde