Microplate reader at 450 nm

Cells were cultivated in 96-well plates after transfection. Afterward, cell counting kit-8 (CCK-8; Solarbio, Beijing, China) solution was applied to incubate cells. Cell viability was examined via the application of microplate reader at 450 nm (Biotek, Winooski, Vermont).
For 5-ethynyl-2′-deoxyuridine (EdU) experiment, EdU Detection Kit (RiboBio, Guangzhou, China) was applied for cell proliferation test basing on the manufacturer’s instructions. EdU-positive cells were captured through utilizing a fluorescence microscope (Leica, Wetzlar, Germany).

Cells in the logarithmic growth phase were made into single-cell suspension with trypsin digestion and counted using a blood cell counting board. BPH-1 cells (4000 or 3000 per well) were inoculated in 96-well plates with six replicate wells for 24 h at 37 °C and 5% CO₂ and then treated with vehicle (0.1% DMSO (v/v)), doxazosin (40 μM), or Cu B (12.5, 25, 50, 100, 200 nM) for 48 h and 72 h. Subsequently, 10 μL of CCK8 reagent was added to each well and incubated for 1–4 h at 37 °C. The optical density (OD) of each well was measured using a microplate reader at 450 nm (BioTek, Winooski, VT, USA). CCK-8 assays were performed in triplicate. The cell viability was calculated as Cell viability (%) = [(A_sample − A_blank)/(A_vehicle − A_blank)] ×100%, where A_sample is the optical density of wells with cells, CCK-8 reagent and Cu B solution, A_blank is the optical density of wells with medium and CCK-8 reagent but no cells, and A_vehicle is the optical density of wells with cells and CCK-8 reagent but no Cu B solution.

Cell viability and proliferation on the scaffolds was assessed by the CCK-8 assay (Boster) and a LIVE/DEAD kit (Sigma-Aldrich). Briefly, a suspension of 1 × 10⁴ BMSCs in 100 μl GM were loaded onto the cubic PLA/HA composite scaffolds. After 1, 3, 5, and 7 days of culturing, viability and proliferation of BMSCs were visualized using LIVE/DEAD kit. Live cells per microscopic field were counted using Image-J software. Furthermore, CCK-8 kit was employed to quantitatively evaluate the cell viability. The optical density (OD) value was read by a microplate reader at 450 nm (Bio Tek Instruments, Winooski, VT, USA).

Spleens from immunized mice were dissected and cell suspensions were obtained. Cells were washed once with RPMI 1640 containing 10% fetal calf serum (heat‐inactivated, endotoxin‐free; FCS, Gibco), streptomycin (100 mg mL⁻¹), and penicillin (100 U mL⁻¹). RBCs were removed by RBC lysis buffer and the remaining cells were washed twice in cold DPBS. Cytokine generation was assessed by culturing splenocytes (10⁷ cells mL⁻¹) in triplicate with HA (10 mg mL⁻¹ or OVA 10 mg mL⁻¹). Control stimuli included RPMI 1640 medium only. Supernatants were harvested after 72 h at 37 °C, 5% CO₂ condition, filtered and stored at −78 °C before use. For quantification of the Th1‐associated cytokine IFN‐γ, ELISA was used following the manufacturer's protocols. Briefly, 50 µL of twofold diluted samples in the blocking buffer were added into the precoated mouse IFN‐γ Microtiter 96‐well plate and incubated with biotinylated IFN‐γ antibody for 2 h at RT. Then, HRP–streptavidin conjugates (100 µL well⁻¹) were added and incubated for 30 min. The reaction termination of 0.18 m sulfuric acid was added. The absorbance was recorded by a microplate reader at 450 nm (Bio‐Tek, USA). The IFN‐γ production of the sample was calibrated against recombinant mouse IFN‐γ standards.