Table 1.

CAPA troubleshooting.

Problem	Cause	Solution
Chloroalkane coupling byproduct	Internal cyclization or side-reaction occurring. We have observed this with some coupling reagents.	Use PyBOP, and avoid HATU or HBTU, when coupling on-resin
Gating removes more than 60% of cells	Cells do not express enough HaloTag-GFP	Select cells with 20 μg/mL puromycin
Cells are lost during aspiration	Aspirating technique is not optimal, or vacuum is too strong	Add a non-filtered pipette tip to the end of the aspirating pipette to reduce surface area of the vacuum; reduce strength of the vacuum pump
Cells do not lift up from bottom of the plate during trypsinization	Low level of protein in the optiMEM inhibits trypsin	Wash with PBS between the final optiMEM wash and trypsinization
Low cell counts	Toxic molecules or handling issues	Use a lower concentration of ct-molecule; handle cells cautiously
Samples measured later in the plate have lower cell counts or more inconsistent results	Cells sitting for too long, not as healthy as those earlier inthe plate	Split up samples into two plates with staggered incubation and read times; alternatively, lower cell counts for each well or increase capillary flow rate to decrease data collection time
Histogram shows a double peak	Cells are clumping	Mix cells more extensively during PBS resuspension; gate for single cells
Curves for the minimum and maximum control samples are overlapping	Background fluorescence is too high and/or maximum fluorescence is not high enough	Adjust gain of red fluorescence detection; lengthen the final wash step; change to a brighter ct-dye