2.8. Steroid Hormone Secretion

The production of testosterone was determined using an enzyme-linked immunosorbent (ELISA) assay. The ELISA method is an immunological reaction that combines the specific reactivity of antigens and antibodies with the efficient catalytic action of enzymes on substrates. TM3 cells were plated into 96-well plates at a density of 4 × 103 cells/well for 24 h. Pre-cultured cells were subsequently exposed to 62.5–2000 µg/mL of Lepidium sativum L. for 24 h and 48 h. After this time period, the cell culture media was aspirated from each well, centrifuged (3000 rpm; 10 min; 4 °C), and supernatants were stored in Eppendorf tubes at −80 °C until steroid determination. The procedure for progesterone and testosterone analyses was carried out according to the manufacturer’s instructions in ELISA kits (progesterone Cat. #{"type":"entrez-nucleotide","attrs":{"text":"K00225","term_id":"174437"}}K00225, testosterone Cat. #{"type":"entrez-nucleotide","attrs":{"text":"K00234","term_id":"176476"}}K00234, Dialab, Wiener Neudorf, Austria). The optical density was measured at a 450-nm wavelength using a microplate reader Multiscan FC. The sensitivities of both steroid hormones are presented in Table 1.

Intra-assay, inter-assay variability, and sensitiveness for the selected steroid hormones.