Table 1 Troubleshooting table

15

The cells are lumpy after plating

Incomplete digestion

Before washing the cells by using PBS, the culture medium should be discarded completely. After cleaning with PBS, aspirate and discard the PBS completely. After discarding trypsin, the cell culture bottle should be placed in the incubator without disturbing the cells

Over-digestion

Incubate strictly according to the protocol timeline to avoid a prolonged digestion period

23

Cells are detached from the surface of the culture flasks

When washing or adding culture medium, the liquid is added directly to the cells

When rinsing cells or adding culture medium, tilt the culture bottle to make the cellular side face up. Add the liquid to the acellular side. When the liquid flows to the bottom of the flask, turn it gently to make the liquid infiltrate the cell surface. This method should be applied to all cell-processing operations

38

The titers of pseudotyped virus are too low

The transfection efficiency was low

Lipofectamine 3000 should be fully mixed with the plasmid in Step 19. The liquids in tubes A and B should be fully mixed in Step 20 so that the liposome can encapsulate the plasmid efficiently

39

The sample is not clear

The serum/plasma sample contains blood lipids or fiber precipitation

After inactivation, serum or plasma samples should be centrifuged at 6,000g for 10 min. Then, the supernatant will be used for follow-up detection

48

The RLUs for the VC are not exactly the same in different runs

Even diluted according to the same TCID₅₀, the RLU may not be exactly the same

Following the recommended dilution fold in the TCID₅₀ macro, it is acceptable as long as the VC/CC value is not <1,000 in the pseudotyped virus neutralization assay

%CV between duplicates is >30%

Inaccuracy of sample addition or dilution

When adding or diluting samples, observe the liquid level in the pipette tips to ensure an identical volume. It is recommended to use a multi-channel pipette to do the serial dilutions