Table 4.

Summary of critical steps of the protocol with possible troubleshooting

Problem	Solution
High variability in readout across triplicate wells	The major source of such variability is unequal number of cells in the different wells. The HEK293 cell is readily detached from the plate during the trypsinization step, but it does not dissociate easily from each other. Ensure that cells form a single-cell suspension at the step of plating. In addition, wrapping the plate in aluminum foil during incubation time in a CO2 incubator will help to maintain even temperature across the plate and as result more even growing.
	Since HEK293 cells detach easily, to prevent the loss of cells during the assay, avoid aspiration of media or plate washing.
Low level of luciferase readout	The aliquot of reporter AAV lost activity or the titer was miscalculated. Take another aliquot or re-titer virus.
	The quality of the HEK293 cell is also very important. Cells should be of low passage and be 50–70% confluent at the beginning of the experiment.
	To increase the signal for serotypes with low infectivity, several approaches can be utilized. (1) Time of incubation can be extended from 24 to 48 h. (2) Multiplicity of infections can be increased. However, make sure that luciferase signal is dose dependent and is not saturated. (3) For many serotypes, pretreatment of HEK293 cell with Compound C together with interleukin-6 and tumor necrosis factor alpha will additionally increase the luciferase readout.
The RLU of MAX luciferase signal is significantly lower than some of the dilutions of the test sample	FBS is used as a diluent and it inhibits AAV infection by itself. Different providers and a lot of FBS should be tested on the ability to affect the AAV infectivity.