Table 2.

Critical Parameters and Troubleshooting

Observation	Cause	Resolution
Not all cells form perfect spheroids	Some cells, such as A549, form spherical shape that can be measured with better accuracy, whereas Calu-3 cells form aggregates of cells.	The measurement of diameters must be optimized for these cells. Ultra-low-attachment plates serve better for cells that form aggregates.
PLA could not be performed on intact spheroids, and the red fluorescence dots could not be visualized using the intact spheroids	Fluorescence light emission from deep within the spheroid did not reach the detector. Poor permeability of antibodies and probes into the intact spheroids may also be a factor.	The spheroids were broken up with vigorous pipetting before PLA.
The direct transfer of intact spheroids to 8-well chamber slides for treatment, and further incubation, led to the growth of the spheroids as monolayer cells	When transferred to the chambers, the spheroids started growing as a 2D cell monolayer because of the completely flat surface of the chamber.	Spheroids were transferred to the bottoms of the wells of 96-well plates for treatment. The assay was carried out on broken spheroids.