TABLE 2.

Troubleshooting table.

Problem	Possible reason	Solution
Step 19: No distinct population of ASCs is present during flow cytometry	Poor or unusual response to vaccine	Use the specified gating strategy to collect the ASCs that are present
Step 22: Negative controls (wells with no cells) on RT/nested PCR have a positive band	Contamination	Plates will probably have to be discarded
Step 25: Negative controls on cloning PCR have a positive band	Contamination	Repeat cloning PCR for contaminated reactions
Step 50: A portion of miniprepped colonies do not contain the insert or there are mutations so that no consensus is found from the four picked colonies	PCR-introduced errors are not uncommon with the large number of PCR cycles required for single-cell PCR	Pick four more colonies from the plate, miniprep and sequence until there are enough sequences to establish a consensus
Step 79: After transfection, the concentration of antibody produced is very low (less than 50 μg ml−1)	Beads have reached capacity or the particular antibody is a ‘poor’ expresser. It is clear that not all mAbs will be expressed in abundance	Run transfection media from which the antibody has been purified on a protein gel. If antibody bands are still present, discontinue the use of that set of beads and purify the supernatant again with a new set. Otherwise, more transfections may be required
	Plates not confluent enough or cells have been passaged for too long	Run transfection media from which the antibody has been purified on a protein gel. If antibody bands are not present, transfect again, checking confluency of plate. Some antibodies do not transfect well; in such cases, transfect eight or more plates instead of four

ASC, antibody-secreting cell.