Table 6 Troubleshooting for the PRNT

1, 2

Staining of monolayer is patchy or uneven

Cells might have been subconfluent on day 1 of the assay

• Ensure that cells are ≥85% confluent and are an even monolayer before setting up on day 1

• Ensure that the correct medium was used to plate the cells; i.e., PRNT/MNA maintenance medium was used, not diluent medium

• Ensure that the incubator parameters are within range for cell maintenance and plating

• Prior to harvesting cells for seeding, ensure that the cells are in logarithmic growth (slightly subconfluent)

Cells might have dried up during the assay setup

• Ensure that there is always residual liquid on the cells

• Ensure that the paper towel inside the incubation box is saturated with water

• Ensure that the box seal is sound; this can sometimes be rectified by greasing the seal with silicone/vacuum grease

Circular hole in monolayer visible in 24-well plates after staining (Fig. 6b)

Cells might have been overconfluent on day 1 of the assay. Following washes and further incubation for 5 d, cells can accumulate in the center of the well and detach

• Check that the calculations for seeding densities are correct

• Ensure that cells are homogeneous and well suspended following trypsinization to enable accurate counting and calculation of cell density

• Ensure that cells are an even monolayer before setting up on day 1

• Reoptimize cell seeding densities if cells are growing at different rates for specific laboratory setups

4, 11

VOC counts are too low

The virus titer might have been reduced during setup if a delay results in virus being held at room temperature or at the neutralization incubation step for too long

• Ensure that the virus is thawed quickly using hand heat only

• Use the virus immediately upon thawing

This effect can be observed if low-absorption plates are not used for the neutralization plate as virus might bind to the surface of the plate

Ensure that the correct low-binding 96-well plates are used for the neutralization plate setup (e.g., Thermo Fisher Scientific, cat. no. 249935)

5

No visible plaques in sample wells where foci are present in the VOC wells of the control plate

Highly neutralizing serum

Retest the serum, starting at a higher starting dilution e.g., 1/320

14, 15

Half-moon-shaped areas on 24-well plates with no or few cells visible after staining (Fig. 6a)

Drying of monolayers during removal of PBS due to removal of too much liquid or length of time taken to replace liquid is too long

Ensure that sufficient volume is left on cells during removal of PBS. If the removal of PBS is taking too long, rock the remaining fluid over the cells to reflood the well and prevent drying

Cell plates are being knocked in transit and not being incubated flat during the adsorption step

Ensure that the plates are incubated flat, particularly during the adsorption step where the volume of liquid is low

14, 17

Damage to cell monolayer visible after staining

Where liquid is removed from the cell monolayer with a plate washer or stripette, the tip can scratch the cells away from the plate

• During these steps, take care not to touch the cell surface with the stripette. The plate should be tilted and the tip of the stripette submerged to just above the base of the plate

• Ensure that the washer aspiration and dispense settings are at their slowest/gentlest levels

• Ensure that the washer nozzle offsets are set so that the nozzles do not touch the base of the plate

• If possible, adjust the nozzle offsets to aspirate and dispense at the extreme edge of each well rather than the center

19

Uneven distribution of plaques with more plaques clustered in the center of the well

Following addition of the virus for the adsorption stage, swirling instead of rocking of plates can cause the virus to accumulate in the center of the well

Ensure that plates are rocked following addition of virus, not swirled

20

Uneven distribution of plaques with clustering of plaques consistently to one side of the well

Plates could have been incubated at an angle during the adsorption step

Ensure that plates are stacked evenly prior to incubation

The incubator might not be level

Adjust the incubator and check the shelves are level

22

Plaques have a large or comet-shaped morphology (Fig. 6c)

Viscosity of CMC can vary between different batches from the supplier

Reoptimize the % (wt/vol) of CMC used in the overlay medium by running a VOC plate with a range of CMC concentrations. The overlay medium should be of a consistency that can be easily transferred by stripette but thick enough to contain the virus to allow a well-defined plaque to form

35, 36

A neutralization graph is not generated by R

The worksheet for analysis was not saved as a .csv file

Ensure that the worksheet is saved as a .csv file

The layout of the .csv file is not as described in Step 35

Ensure that the worksheet is set out as described in Step 35