Imark plate spectrophotometer

To determine the Iba1, CD86, IL-6, and IL-1β protein concentrations within the hippocampus, an enzyme-linked immunosorbent assay (ELISA) was used. Rats were anesthetized with isoflurane using rodent anesthesia vaporizer (VetFlo, Kent Scientific Corporation, Torrington, CT, USA) and each hippocampus was quickly extracted, frozen in liquid nitrogen, and stored at a temperature of −70 °C. The following ELISA kits were used for quantification of Iba1 (NBP2-66675, NovusBio), CD86 (RAB0887-1KT, Sigma-Aldrich, St. Louis, MO, USA), IL-6 (ERA32RB, Invitrogen), and IL-1beta (BMS630, Invitrogen) according to the manufacturer’s instructions. For analysis, we used both right and left hippocampi. The homogenization buffer consisted of 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, and 0.5% sodium deoxycholate, with a protease inhibitor cocktail (cOmplete, Sigma-Aldrich, St. Louis, MO, USA). The BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used for determining protein concentration. The optical density was measured in an iMark plate spectrophotometer (Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm.

A549, HT-29, and HCT116 cells were seeded in 96-well plates at a density of 1 × 10⁴ per well in 100 μl culture medium and incubated for 24 h in humidified atmosphere of 5% CO₂ (New Brunswick, Eppendorf, Germany) at 37°C. The culture medium was aspirated and 100 μl of fresh serum free medium supplemented with TRAIL or DR5-B was added to the wells. In the case of Jurkat, cells were harvested, washed with serum-free medium and plated in 96-well plates (5 × 10⁴ cells per well) in 100 μl of culture medium without serum and 50 μl of TRAIL or DR5-B solutions at the appropriate concentrations were added to each well. The cells were incubated for 24 h, 10 μl of water soluble tetrazolium salts reagent (WST-1) (Sigma-Aldrich, St. Louis, MO, United States) was added to each well and incubation was continued for another 2 h at 37°C. The WST-1 assay is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Viable cells have a high activity of mitochondrial dehydrogenases, which leads to the formation of the dye formazan. The optical density of the wells was measured using an iMark plate spectrophotometer (Bio-Rad, United States) at a wavelength of 450 nm with background subtraction at 655 nm.

To determine the concentration of cytokines in the mouse hippocampus, the enzyme-linked immunosorbent assay (ELISA) was used. The animals were anesthetized with isoflurane and the hippocampi were quickly removed, frozen in liquid nitrogen, and stored at −80 °C. Both the right and left hippocampus were used for analysis. The sample homogenization was performed using a homogenizing buffer consisting of 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA and 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate with a cocktail of protease inhibitors (cOmplete™, Sigma-Aldrich). The obtained homogenates were incubated on ice for 15 min, centrifuged (16,000× g, 30 min, +4 °C), and supernatants were collected. For protein concentration analysis, the BCA kit (Pierce, Rockford, IL, USA) was used. ELISA kits were used for the detection of TNF-α (ab208348) and IL-6 (ab46100), all from Abcam. The absorbance at 450 nm was measured using an iMark plate spectrophotometer (Bio-Rad, Hercules, CA, USA).

Animals were deeply anesthetized with isoflurane using a veterinary vaporizer (VetFlo™, Kent Scientific Corporation, Torrington, CT, USA). The brain was quickly removed from the skull, and the ipsi- and contralateral hippocampi were isolated separately, frozen in liquid nitrogen, and placed in a freezer at t = −70 °C for storage. For analysis, hippocampi were homogenized in a solution of the following composition: 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA and 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and a mixture of protease inhibitors (cOmplete™, Sigma-Aldrich, Bellefonte, PA, USA). The resulting homogenate was incubated on ice for 15 min, centrifuged (16,000× g, 30 min, +4 °C), and the supernatant was collected. We used a Superoxide Dismutase Activity Assay Kit for the analysis according to the manufacturer’s instructions (CS0009-1KT, ™, Sigma-Aldrich, Bellefonte, PA, USA). Absorbance was measured at 450 nm using an iMark plate spectrophotometer (Bio-Rad, Hercules, CA, USA). A BCA kit (Pierce, Rockford, IL, USA) was used to measure protein concentration.

A549 cells were cultured in DMEM nutrient medium supplemented with 10% fetal bovine serum. HT-29 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were seeded in 96-well plates at a density of 1 × 10⁴ per well in 100 μL culture medium and incubated for 24 h in humidified atmosphere of 5% CO₂ in air (New Brunswick, Eppendorf, Germany) at 37 °C. Culture medium was aspirated and 100 μL of fresh serum free medium supplemented with TRAIL variants and chemotherapeutic drugs was added to wells. After 24 h incubation, 10 µl WST-1 reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and the plates were kept for 2 h at 37 °C. The optical density of the wells was measured using an iMark plate spectrophotometer (Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm with background subtraction at 655 nm.