Safire 5 microplate reader

The alamar blue^® assay to measure cell proliferation (ThermoFisher Scientific Inc., Waltham, MA, USA) was performed according to manufacturer’s instructions. Primary osteoblast cells in passage 3 or 4 (25,000 cells per well in 200 µL medium) were seeded into sterile flat bottom 96-well cell culture plates (Greiner bio-one, Cellstar) and cultivated for 24 h before adding the various β-TCP-based bone substitutes to obtain a final concentration of 100 μg/mL. The control was cells without the addition of substitutes. All experiments were carried out in triplicate and three different osteoblast donors. Proliferation of cells was analyzed at various time points up to 48 h by the addition of alamar blue for 1 hr after which 100 µL was removed for analysis. A multi-mode reader (λex = 530 nm, λem = 590 nm; Tecan Safire 5 microplate reader, Männedorf, Switzerland) was used to measure the fluorescence. The fluorescence values were normalized against the control seeded at the same time onto cell culture plastic (time 0). The proliferation of osteoblasts under the different conditions were compared to the control cells not exposed to substances which were set to 1 at the time of seeding (0 time). Thus, values lower than 1 with time indicate decreasing amounts of viable cells (toxicity of test compound) and values greater than 1 indicate increasing numbers of viable cells (proliferation).

Cell death was measured using a lactate dehydrogenase (LDH) assay based on the detection of the LDH released from injured cells. Mechanistically, LDH can oxidize lactate to generate NADH, which in turn reacts with INT, determined as a yellow coloured substrate with maximum absorbance at 450 nm (Beyotime Company, Jiangsu, China). The 6-week ALI cultures were infected at a multiplicity of infection (MOI) of 1, 10 and 100 per M. ovipneumoniae cell through the application of bacterial cells on the apical surface of cultures for 24 h and then incubated with LDH staining solution at 37 °C for 1 h. Then, the supernatant was used to examine the relative LDH activity using a Tecan Safire 5 microplate reader (450 nm).

The concentration of ROS was fluorometrically monitored using DCFH-DA. The cells were treated with MEK inhibitor PD980025 (Sigma, USA) for 24 h prior to M. ovipneumoniae infection at an MOI of 100 through the application of the bacterial cells on the apical surface, followed by incubation at 37 °C overnight in 24-well plates. After the membranes were washed three times with PBS, DCFH-DA was diluted in fresh phenol red-free DMEM to a final concentration of 5 μM and incubated with the cells at 37 °C for 20 min. The chemicals were then removed, and the cells were washed three times. Relative ROS units were determined using a Tecan Safire 5 microplate reader (λex 485 nm and λem 530 nm). Changes in the ROS concentration were expressed as a percentage over the control.