M1000 plate reader

Before seeding, cells were washed 3x with 5 mL DMEM/F12. Then, 20,000 cells were seeded per well into 96 well plates containing 200 μL DMEM/F12 with 50 ng/mL EGF or vehicle (0.1% [w/v] BSA in PBS). CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) was then performed per manufacturer's instructions. Absorbance was read with a Tecan M1000 plate reader (Tecan Group Ltd., Mannedorf, Switzerland). Background absorbance (culture media without cells) was subtracted, and then values were normalized to day 0.

Total lung collagen content was estimated using hydroxyproline assay as described previously (Tamo et al., 2018 (link)). Frozen middle lung lobes were weighted and homogenized. The homogenate was treated with 10% trichloroacetic acid, hydrolyzed with 6 M HCl (overnight, 110°C) and adjusted to pH 7.0 with NaOH. Oxidation was initiated by incubation with 1 ml of chloramine T-reagent (20 min at RT) and stopped by addition of 1 ml of 3.15 M HClO₄. After incubation with Ehrlich reagent (p-dimethylaminobenzaldehyde added to methyl cellosolve) for 20 min at 55–65°C, absorbance was measured at 557 nm using Tecan M1000 plate reader (Tecan, AG, Männedorf, Switzerland). A standard curve was generated using known concentrations of hydroxyproline.

To determine the level of binding of LB-MSN-OVA on the microneedle arrays, DOPE-LR was added to the lipids when the LB-MSN-OVA were prepared. The top of the microneedle arrays was incubated with LB-MSN-OVA (50 μl) with a concentration of 0.1, 0.5 and 1 mg/mL in EDTA buffer (1 mM, pH 5.8) for 2 h at room temperature. The microneedles were then washed with coating buffer (450 μl) and the solution was kept for measurement. The binding efficiency of LB-MSN-OVA was determined by comparing the DOPE-LR concentration in the coating solution before and after coating by using a Tecan M1000 plate reader (Excitation wavelength = 575 nm and Emission wavelength = 590 nm). The structure, geometry and the surface morphology of the LB-MSN-OVA coated pH-sensitive microneedle arrays were examined by scanning electron microscopy (SEM) in a FEI NOVA nanoSEM 200 (Hillsboro, OR). The LB-MSN-OVA coated on microneedle arrays were also visualized by Nikon D-Eclipse C1 confocal laser scanning microscope (CLSM, Tokyo, Japan) with a depth resolution of 5 μm/step, equipped with a 10 × Plan Apo objective. The x and y resolution was 2.5 μm. An argon laser (488 nm) was used to visualize OVA-AF488 with a 530/55 emission filter and a diode-pumped solid-state laser (561 nm) with a 590/55 emission filter was used to visualize DOPE-LR.

Human placental secreted alkaline phosphatase (SEAP) concentration in the cell culture medium was determined by means of light‐absorbance time‐course measurement.^[73] For this, 20 µL of culture supernatant was mixed with 80 µL of ddH₂O and heat‐inactivated for 30 min at 65 °C. Then, 80 µL of 2x SEAP buffer (20 × 10⁻³m homoarginine, 1 × 10⁻³m MgCl₂, 21% (v/v) diethanolamine, pH 9.8) and 20 µL of 120 × 10⁻³m para‐nitrophenyl phosphate (Fisher Scientific Acros Organics, Geel, Belgium, cat. no. 128860100) solution in 2x SEAP buffer were added to each well and the absorbance at 405 nm was measured at 37 °C using a Tecan M1000 plate reader (Tecan Group Ltd., Maennedorf, Switzerland). SEAP concentrations in serum were quantified using a chemiluminescence‐based assay (Roche Diagnostics, cat. no. 11779842001). In brief, 50 µL of heat‐inactivated serum (30 min, 65 °C), was centrifuged for 30 s at 5000x g and transferred to a well of a 96‐well plate containing 50 mL of inactivation buffer. The plate was incubated for 10 min at room temperature (21 °C), then 50 µL of substrate reagent was added to each well, and incubation was continued at r.t. for 10 min. Luminescence was measured using a Tecan M1000 plate reader and concentrations were calculated from a standard curve.

For a typical trial, a 7.5 μM peptide solution was incubated in the absence or presence of the appropriate concentration of Cts L (30.3 nM), Cts V (20.5 nM), Cts K (42.6 nM), Cts S (21.6 nM), or Cts B (37.6 nM) in 100 mM sodium acetate, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, and pH 5.5 buffer at 27 °C. The fluorescence was monitored as a function of time at 390 nm with an excitation wavelength of 325 nm using a Tecan M1000 plate reader. Three independent trials were conducted for each assay to ensure reproducibility. More details of the assays, along with the raw data and analysis, are included in the ESI.†