Ascent microplate reader

To evaluate the levels of corticosterone and DHEA, tail blood was collected under two conditions: home cage as baseline and 30 min following exposure to the elevated plus maze to evaluate HPA axis response. Blood samples were collected only when the females were in the proestrous or estrous stage of the estrous cycle. To avoid the circadian peak and the diurnal variation in corticosterone, samples were collected between 14:30 h and 16:00 h in heparin-coated capillaries (Roth, TX80.1), and then centrifuged at 3000 g for 15 min. The plasma was collected and stored at −80 °C until the assay was performed. Samples were diluted in assay buffer and analyzed according to the manufacturer’s instructions (Enzo life sciences; corticosterone Elisa kit: ADI-900-097; DHEA Elisa kit: ADI-900-093).
To measure progesterone and 17β-estradiol concentrations, tail blood was collected in heparin-coated capillaries as well. Hormones concentration was determined by ELISA (Demeditec Diagnostics; estradiol Elisa kit: DE2693; Progesterone Elisa kit: DE1561) according to the manufacturer’s instructions. The absorbance was read at 405 nm using the MultisKan Ascent microplate reader and the values were calculated based on the standard curve.

Coating wells with COL1 (rat tail; Roche Diagnostics GmbH, Mannheim, Germany) was performed at 10 μg/cm², coating volume 30 μL per well of a 96-well plate, as described previously [4 (link)]. Plates were placed under laminar air flow at room temperature until complete evaporation of liquid and washed with 50 μL DPBS/well. A total of 30,000 cells were cultured in 160 µL medium on COL1-coated and uncoated surfaces. Non-specific binding sites were saturated with 50 µL of a 1% BSA solution.
After the defined incubation times, each well was aspirated, washed carefully with 100 µL of DPBS, and treated with 50 µL of a paraformaldehyde solution (4%) for 30 min. Staining of the cells was performed with 50 µL methylene blue solution (0.1% in H₂O). Afterwards, wells were washed three times with 200 µL of deionized water, and 100 µL of HCl solution (0.1 M) was added to dissolve the dye. Absorbance was measured in a Multiskan Ascent microplate reader at a wavelength of 630 nm.