Taxol equivalent

To assess the effect of drugs on CAR T cell cytotoxicity, cancer cells were plated at 5,000/well concentration in 96 well plates in triplicate. Next day, cells were treated with drugs (1-Methyl-d-tryptophan (1-MT, #452483, Sigma), indomethacin (#17378, Sigma), gemcitabine hydrochloride (#G6423, Sigma), paclitaxel (Taxol equivalent, #P3456, Invitrogen) and 5-Fluorouracil (#F6627, Sigma)) at indicated concentrations for 24 h. After 24 h, the drug was removed, cells were washed and further incubated with mock or CAR T cells at T:E ratio of 1:10 for 72 h. Thereafter, the percentage of surviving cancer cells were measured using the MTT assay. For Gal-9 and PD-1 blocking experiments, cancer cells were plated at 10,000 cells/well concentration in 96 well plates in triplicate. After 24 h, mock or CAR T cells were added at T:E ratio of 1:10. 0.1, 1, and 10 μg/mL of Gal-9 (clone 9M1-3, BioLegend, San Diego, CA, USA) or PD-1 blocking Ab (clone EH12.2H7, BioLegend) was added to the media and incubated at 37 °C for 72 h. Survival was measured using the MTT assay.

For the present study, we used commercial bovine brain tubulin (catalog No T238, Cytoskeleton, Denver, CO, USA). Tubulin was polymerized with a “hybrid” protocol in either the presence or absence of Paclitaxel (Taxol Equivalent, Invitrogen™, P3456), following general guidelines from the Mitchinson Lab’s online protocols (http://mitchison.med.harvard.edu/protocols/). Briefly, all reactions were conducted in BRB80 solution containing 80 mM PIPES (1,4-piperazinediethanesulfonic acid), 1 mM MgCl₂, 1 mM EGTA, and pH 6.8 with KOH. MT sheets were obtained as previously reported 7 (link).
In the experiments where MTs were polymerized in the presence of Paclitaxel (stock solution, 10 mM), the drug was initially prepared as per the manufacturer's recommendation and diluted to obtain solutions between 2 and 200 mM, which were added sequentially every 5 min to the tubulin suspension as a dilution 1:10 in BRB80, DTT and GTP.

Paclitaxel was used and administered into culture media in accordance with the treatment protocol (Taxol equivalent, Invitrogen, cat No. P3456-5 mg, Schwerte, Germany). Dimethyl sulfoxide (DMSO, Sigma–Aldrich, cat. No. D4540-500 mL, Lyon, France) was used to dissolve paclitaxel to obtain stock solutions of 1 mM and stored at −20 °C.