2.2. PBMC Cytotoxicity Assay

Isolated PBMCs at a concentration of 1 × 10⁵ cells/mL were cultured with CB[6], CB[7] and CB[8] in a culture medium RPMI-1640, supplemented with 50 mg/mL of gentamicin, 25 mg/mL of tienam and 10% FCS in a 96-well plate (TPP, Trasadingen, Switzerland) for 72 h in a humidified atmosphere of 5% CO₂. CB[6] and CB[7] were added at concentrations of 0.3, 0.5 and 1 mM, while CB[8] was added at 0.01 mM due to the low solubility of this compound in mediums. The wells were duplicated. After cultivation cells were collected and washed with PBS, WST assay reagents were added to the cell culture media and then incubated for 4 h. The cytotoxicity of CB[n] was analyzed by the amount of formazan dye produced by measuring the absorbance at 450 nm.

PBMCs at a concentration of 1 × 10⁵ cells/mL were cultured with CB[6,7,8] at the same concentrations and conditions as the WST assay for 24 h to release LDH into the cell culture medium. To perform the assay for the cellular cytotoxicity of CB[n], the reaction mixture was added to the wells of the cell culture medium. After 30 min of incubation with lysis buffer and 30 min of incubation with assay buffer with protection from light, the reaction was stopped by adding a stop solution and the absorbance was measured using a microplate reader Infinite F50 (Tecan, Grödig, Austria) at 490 nm. The average absorbance of each triplicate set of wells was calculated and the background control value was subtracted. The percent cytotoxicity was calculated with the following equation:

A: Test Substance, B: High Control, C: Low Control.