Sinergymx

Freshly collected cell supernatants following stimulation were used for the end-point lactate dehydrogenase (LDH) activity measurement assay according to the manufacturer´s instructions (LDH Cytotoxicity Assay, Roche, Basel, Switzerland). On the cells, we performed XTT test to determine their viability/metabolic activity. For XTT assay, we used DMEM without phenol red and added to it a solution of tetrazolium salt (XTT) and phenazine methosulphate (PMS). Upon colour development we measured absorbance at 490 nm using a multiplate reader SinergyMx (BioTek, Winooski, VT, USA).

CAR-T effector cells were seeded at 1 × 10⁵ cells per well in a 96-well plate and co-incubated with Raji as the target cells at an E:T ratio of 10:1 or with K562, K562-CD19⁺, K562-CD20⁺, or K562-CD19⁺/CD20⁺ as the target cells at an E:T ratio of 5:1 for 48 h. Human IL-2 cytokine concentration in the culture supernatant was detected using an IL-2 uncoated ELISA Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Washing between the incubation steps was performed using a HydroSpeed™ plate washer (Tecan). A multiplate reader SinergyMx (BioTek, Winooski, VT, USA) was used to measure endpoint absorbance.

IL-1β was detected in cell supernatants using mouse and human IL-1 beta uncoated ELISA Kit according to the manufacturer’s instructions (Thermofisher Scientific, Waltham, MA, USA). Multiplate reader SinergyMx (BioTek, Winooski, VT, USA) was used to measure absorbance.

Cytotoxicity of DON, ZEA and their combinations was assessed by the tetrazolium-based (MTS) assay following the producer’s guides (Promega) with minor changes [47 (link)]. HepG2 cells were seeded into 96-well plate. After 24 h, cells were exposed to DON (0.25, 0.5, 1, 2, 4, 8 and 16 μM), ZEA (1.25, 2.5, 5, 10, 20, 40 and 80 μM) or to DON and ZEA (at concentrations: 1 μM DON + 5 μM ZEA, 1 μM DON + 10 μM ZEA, 1 μM DON + 20 μM ZEA) for 24 h. At the end of treatment period, we added 20% freshly prepared mixture of PMS:MTS solution (1:20) to each well and incubated for further 3 h. After the incubation period, absorbance was read at 490 nm on microplate reader (Sinergy MX, BioTek, Winooski, VT, USA). The experiment was conducted as five replicates per treatment point and repeated in three independent biological replicates. For each experiment we included negative control (cell medium), mycotoxin solvent control (cells exposed to 0.1% DMSO) and positive control (30 μg/mL ET).
The difference in cell viability among treated groups and solvent control group was analysed by the one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test with the program GraphPad Prism V8 (GraphPad Software, San Diego, CA, USA). The level of statistical significance was set at p < 0.05.

Viability of HepG2 cells was determined after 24 and 72 h of exposure to pure cyanotoxins and their combinations with the MTS tetrazolium reduction assay in accordance with the manufacturer’s protocol. At the end of the incubation, 20% of fresh MTS: PMS mixture (20:1) was added to treated cells and incubated for 3 h. The viability of cells was evaluated by measuring the optical density (490 nm) using a spectrofluorimeter (Sinergy Mx, BioTek, Winooski, VT, USA), which correlates to the amount of viable cells that are able to reduce tetrazolium to formazan. Three independent experiments were done, for each treatment time in five replicates per treatment point. Statistical analysis among treatments and solvent control was performed by One-way analysis of variance (Dunnett’s Multiple Comparison Test) with GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) (* p < 0.05).