TROUBLESHOOTING

Step	Problem	Possible Reason(s)	Possible Solution(s)
33	CAR does not express	Improper protein folding and/or trafficking of receptor to cell membrane	Test multiple scFv sequences or ligand-binding domains (Fig. 2a).
48	CAR-T cell does not signal	Receptor affinity too low to transduce signal upon ligand binding.	Test different scFv sequences or ligand-binding domains (Fig. 2b). Include positive control to test whether CAR responds to immobilized antigen.
		Poorly signaling CAR	Control for ability of CAR to signal in a traditional manner using an immobilized antigen target (Fig. 2c).
		Poor T-cell product	Control for ability of T cell to produce an effector response using PMA/ionomycin stimulation (Fig. 2c).
8–33	Cell line with low viability	Poor donor	Test primary cells from multiple donors.
		Improper maintenance of T-cell cultures	Maintain cultures at 1 × 106 cells per mL; supplement cultures with 50 U/mL IL-2 and 1 ng/mL IL-15 every 2–3 days.
34–42	Poor yield from cell sorting	Poor transduction efficiency	Make sure transduction efficiency is sufficiently high (>20–30%) prior to sort (Supplementary Fig. 2).
		Positively transduced cells inadequately labeled	Increase labeling time with antibody.
51A	Poor cell viability	Brefeldin A toxicity	Reduce incubation period in Brefeldin A. Depending on the exact measurement desired in the experiment, this can be performed by either shortening the incubation in 51Aiii to 4–12 hours, or initiating the experiment without Brefeldin A and adding Brefeldin A to cells 4–12 hours prior to completing the incubation.
73	Cells touching when attempting to image without cell-cell contacts	Cells are too dense	Test various cell densities.
74	Cells flow out of view after adding ligand to well when imaging	Addition of ligand causes too much disturbance to wells	Use higher concentration of ligand at smaller volume; apply ligand further away from region being imaged.