Mtt assay

Cell viability was assessed by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Millipore, Poznan, Poland), which is based on the detection of the mitochondrial activity. First, cells were trypsynized and seeded on 96-well plate at density 10⁴ cells/well. After 24 h, the exposure with ST2C solution was initiated during 1, 2, 12, and 24 h. Post the exposition MTT assay was performed according to the manufacturer protocol, and formazan crystals were dissolved in acidified isopropanol. The absorbance values were measured at 570 nm by the multiplate reader (Glomax, Promega, GmbH, Mannheim, Germany). All the results were calculated as the percentage of viable cells in the relation to untreated control cells.

To test whether IgE affects T-cell survival and proliferation, we seeded different numbers of CD4⁺ or CD8⁺ T cells (2.5 × 10⁵ to 1 × 10⁶ per well) on a 96-well flat-bottomed tissue culture plate containing both anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) antibodies. Cells were treated with murine IgE (50 μg/ml; Sigma-Aldrich) for 3 days. MTT assay was then used to detect cell survival, according to the manufacturer's instructions (Millipore). Medium absorbance was read at 570 nm. ELISA (BD Biosciences) was then used to determine IL12 levels from T-cell culture media.
To detect cytokine production in CD4⁺ and CD8⁺ T cells, T cells (2.5 × 10⁶/ml) were cultured in a complete medium (RPMI 1640 medium and 10% fetal bovine serum) in anti-CD3 mAb (1 μg/ml, BD Pharmingen) pre-coated plates in the presence or absence of different doses of IgE (0–100 μg/ml; Sigma-Aldrich) for 2 days. Cells were collected for total RNA preparation using the Qiagen RNA isolation kit for RT-PCR (Bio-Rad, Hercules, CA) to determine mRNA levels of IL6, IFN-γ, and IL10. Cell culture media were collected to measure secreted cytokines by ELISA, according to the manufacturer's instructions (BD Biosciences).

Cells were plated in 24-well plates at a density of 1×10⁴/ml in 100 ml of steroid-free medium per well. Three days later, cells were treated with steroid-free medium containing 25 nM Δ4A and different concentrations of letrozole. The medium was changed every 3 days, and the cells were counted 9 days later using the MTT assay (Millipore, Billerica, MA, USA). Final absorbance was measured in triplicate with a microplate reader at 570 nM (Bio-Rad680). The results were expressed as a percentage of the cell number compared with the vehicle-treated wells (control).

The viability of TUFM-deficient cells was validated by the MTT assay (Millipore) in accordance with the manufacturer’s instructions.

Lymphocytes from immunized and control (vehicle and ova-treated) mice were isolated after second immunization. A total of 2 × 10⁶ splenocytes were pre-stimulated for 5 days in the presence of 5 × 10⁵ irradiated (3-Gy), naïve splenocytes, acting as antigen presenting cells, and 10 μg/mL of each peptide. Pre-stimulated splenocytes were tested for GL261-specific cytotoxicity using 10:1, 20:1 and 40:1 effector:target (E:T) ratios. The cytotoxic MTT assay was performed according to the manufacturer’s instructions (Millipore). For proliferation assay, 2 × 10⁶ splenocytes were primed for 4 days in the presence of 5 × 10⁵ naïve splenocytes that had received 3 Gy irradiation, 10 μg/mL of IDH1 mutated or OVA peptides and 10 U/ml of IL-2. After pre-stimulation, 5 × 10⁵ splenocytes were incubated for 20 h in the presence of single peptides (10 μg/ml) and 10 U/ml IL-2 and tested for their ability to proliferate using MTT Reagent (Millipore). The data are expressed as the percentage of proliferation, calculated according to the following equation: (OD stimulated splenocytes – OD splenocytes without peptide)/OD stimulated splenocytes × 100.