Ldh cytotoxicity assay

An LDH cytotoxicity assay (Sigma-Aldrich 11644793001) to measure lysis was conducted according to the manufacturer’s instructions. Briefly, 2.0×10⁴ HeLa cells were seeded per well in a 96-well plate overnight. For cholesterol depletion, cells were incubated with 4 mg ml⁻¹ methyl-ß-cyclodextrin (MßCD) for 1 h and washed with PBS. Cells were incubated in appropriate dilutions of toxin in fresh medium for 1.5 h. 100 µl of the supernatant was transferred to a new 96-well plate and 100 µl of reaction mixture consisting of dye solution and catalyst (prepared according to the manufacturer’s protocol) was added. Plates were incubated in the dark for 30 min. Absorbance of samples was measured at 492 nm.

For the specific lysis assays, luciferase-expressing tumor targets (1 × 10⁴ cells) were co-cultured with varying amounts of p32 CAR T, Sp6 CAR T or UT T cells, for 18 h. Luminescence was measured using a IVIS lumina IIITM system (Perkin Elmer) immediately upon addition of Luciferin (D-Luciferin Firefly, potassium salt Cat No. LUCK250, GOLDBIO, Gold Biotechnology). Other cytotoxicity assays included LDH cytotoxicity assay (Sigma-Aldrich, Cat. No. 11644793001), and co-culture of GFP positive target cells with varying amounts of p32 CAR T, Sp6 CAR T or UT T cells for 3–5 days. Cells were collected, stained and analyzed by flow cytometry.