Toxilight non destructive cytotoxicity bioassay kit

The cytotoxicity of the CAR-T cells was performed with ToxiLight™ non-destructive cytotoxicity bioassay kit (Lonza) and as manufacturer’s instruction described [29 (link)]. Briefly, the MCL cell lines or primary MCL patient samples (lymphoma % ranged from 73.7% to 98.7%) were co-cultured with 74bbz CAR-T cells or untransduced T cell control (UTT) cells at E:T ratio of 5:1 for 24 h. At 24 h, the wells for maximum lysis were added with 100% Lysis Buffer (Lonza) for 10 min at room temperature. The volume in other wells were adjusted with the provided Tris AC buffer. The cell supernatant from each well was harvested and reacted with the provided substrate for 5 min before the plates were read for bioluminescence with a Synergy HT microplate reader (Biotek, VT).

AK assay was performed using the ToxiLight™ non-destructive cytotoxicity bioassay kit (Lonza, Switzerland). The basolateral media were collected on Days 1, 2, and 3, diluted with five volumes of AK detection reagent, allowed to incubate for 5 min, and then read in an Infinite 200® PRO instrument (TECAN, Switzerland). The results of the ToxiLight™ non-destructive cytotoxicity bioassay kit were represented as delta relative luminescence units (ΔRLUs). The following equation explains how ΔRLU is calculated: ΔRLU = L_P − L_A, where L_P is a unit obtained in the presence of CB and EGF, and L_A is a unit obtained in the absence of CB and EGF.

Polycaprolactone (PCL,
Mn 80,000), fetal bovine serum (FBS), mercaptoethanol, penicillin/
streptomycin, 70% ethanol, T75 tissue culture flask, 24-well tissue
culture plates, papain, dexamethasone-cyclodextrin DEX-CYD, mass ratio
65:1000 = dex:(dex + cyd), granulocyte-macrophage colony-stimulating
factor (GM-CSF), lipopolysaccharide (LPS) from Escherichia
coli, macrophage colony-stimulating factor (M-CSF),
Interleukin 4 (IL-4), methanol, dichloromethane (DCM), cetylpyridinium
chloride (CPC), Alizarin Red, nonessential amino acids, β-glycerophosphate,
and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich,
U.K. THP-1 cell line (ATCC no. TIB-202), phorbol 12-myristate-13-acetate
(PMA), Quanti-iTTM Picogreen kit, mouse anti-human calprotectin antibody,
Rhodamine red goat anti-mouse IgG (H + L) secondary Ab, Alexa Flour
488 goat anti-rabbit IgG (H + L) secondary Ab, and DAPI and RPMI-1640
were purchased from Fisher Scientific, U.K. α-MEM (BE12–169F),
and ToxiLight nondestructive cytotoxicity bioassay kit were purchased
from Lonza, U.K., and rabbit anti-human MR Ab from Abcam-U.K.

Assessment of cytotoxicity or cell stress was analyzed by estimation of adenylate kinase (AK) [18 (link)] release from the endothelial cells (EPI-EC; HBMEC) and astrocytes (EPI-Astro; HA) subsequent to DAPK inhibition. Cells were seeded in multiple 12-well plates at a density of 1 × 10⁵ cells per well and left overnight to adhere. Then DAPK inhibitor (100 μM) was prepared in the respective media and incubated for 24 h. Cell morphology of the brain endothelial and astrocytes were periodically monitored. Media samples were collected before and after the treatment with DAPK inhibitor. The detection was performed with the use of ToxiLight™ Non-destructive Cytotoxicity BioAssay Kit (Lonza, NJ, USA) following manufacturer instructions. The detection reagent was added to the samples, and photon emission indicating the presence of ATP was recorded by spectrophotometer (BioTek, Synergy HT, USA).