Luna 2

BT549 cells were divided into 24-well plates with 1000 cells per well. After allowing the cells to adhere, cells were treated with CHNQD-00824 at a concentration of 5 μM. The DMSO-treated group was used as a negative control. Three wells of each treatment group were digested every 24 h and counted separately using an Automated Cell Counter (LUNA-II, logos biosystems). Counts were performed for 7 consecutive days. A cell proliferation curve was drawn to compare the cell proliferation rate.

Adherent THP-1 cells were treated with ceramics as per the protocol above. Cells were then treated with 1 mL Accutase (Sigma-Aldrich) and incubated for 15 min in order to detach the cells from the tissue culture plate. Following incubation, the cells were collected in Eppendorf tubes and centrifuged at 5500×g for 5 min. The cell supernatant was removed, and the cells were resuspended in 200 μL complete RPMI-1640 medium. 10 μL of cell suspension was then mixed with 10 μL trypan blue (Sigma-Aldrich) and mixed gently. 10 μL of this mixed solution was then used on a Luna cell counting slide in conjunction with the Luna II (Logos Biosystems, Seoul, South Korea) fully automated cell counter in order to calculate percentage viability. Additional THP-1 cells were seeded at a density of 50,000 cells per well of a 96-well tissue culture plate and treated in accordance with the protocol above. The XTT Cell Proliferation Kit II (Sigma-Aldrich) was used according to the manufacturer’s instructions and absorbance read using a BioTek Synergy HT microplate reader at 450 nm.

ARPE-19 cells were obtained from the Cell Culture Collection of the Koltzov Institute of Developmental Biology of Russian Academy of Sciences. RPE cell suspension at a concentration of 0.5 × 10⁶ (cells/dishes) was cultured in a supplemented culture media DMEM/F12 containing 10% of embryonic veal serum, 2 mM L-glutamine, and antibiotic–antimycotic (streptomycin, amphotericin B, penicillin) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Cultivation was carried out in Petri dishes (d = 35 mm) under standard conditions (37 °C, 5% CO₂) for 3 days. Thereafter, a change in complete nutrient medium was carried out. When the cells reached 70% of confluence, the nutrient medium was completely removed and 500 µL of accutase solution (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added, followed by incubation for 10 min. Then, the complete nutrient medium was added to the cell suspension to dilute the accutase 10 times. The resulting cell suspension was centrifuged (1100 rpm, 5 min, 37 °C). After centrifugation, the supernatant was removed and 1 mL of complete nutrient medium was added. To count the cells, the resulting suspension was placed in 10 µL disposable slides. An automatic counter of cells, Luna II (Logos biosystems, Anyang, Korea), was used.

In the other set of mice, bronchoalveolar lavage was performed as previously described by our group [16 (link)]. Briefly, lungs were washed three times with 0.7 mL of sterile saline. Total cells were counted using an automated cell counter (LUNA-II, Logos Biosystems, France) and the BAL fluid was centrifuged at 1000× g for 10 min. The supernatant was stored at −80 °C until further analyses and the pellet was resuspended in PBS for differential cell counts. A volume of 100 µL of resuspended cells adjusted to 0.75 × 10⁶ cells/mL were spun at 450 rpm for 6 min (Cytospin™4 cytocentrifuge, Thermo Fisher Scientific, Waltham, MA, USA) onto microscope slides. Slides were air-dried, fixed in methanol for 10 min and stained with May–Grünwald (Química Clínica Aplicada S.A, Tarragona, Spain) for 5 min and 30% Giemsa (Merck, Darmstadt, Germany) for 15 min. Counts of macrophages, neutrophils, eosinophils, basophils and lymphocytes were carried out in a total of 400 cells from each sample in a blinded manner.