Ab37355

To enable the biorecognition assays, the gold surface is modified with detection proteins using noncovalent physicochemical adsorption. First, protein A/G (recombinant fusion protein with protein A- and protein G-binding sites) solution (500 μg ml⁻¹) in 10 mM acetate buffer (pH 4) with 0.5% glycerol is deposited on the clean Au sensor in a microarray format. A non-contact, low-volume liquid dispenser (sciFLEXARRAYER S3, Scienion, Dortmund, Germany) is used to create microarrays of protein A/G droplets (150 pl) with 100 μm diameter and 250-μm period. After 1 h incubation, the chips are washed in phosphate-buffered saline (PBS, pH 7.4) for 5 min under constant agitation to remove excess proteins. The remaining area on the bare Au surface is blocked by incubating the whole chip in 1 % v/v bovine serum albumin (BSA) in PBS. Then, the chip is sequentially incubated in mouse IgG solution (Abcam-ab37355, Cambridge, UK, concentration 200 μg ml⁻¹) and its matching goat anti-mouse IgG solution (Sigma-Aldrich-M2650, St Louis, MO, USA), each step followed by PBS washing. Wet media measurements are performed in PBS buffer, whereas for dry measurements, chips are rinsed in deionized water to remove the salt residuals and dried with N₂ after each incubation step.

Cells collected from BALF were incubated in 100 μl 1×RBC lysis buffer (BioLegend) on ice for 10 min, and then quenched by adding 1 ml PBS. Cells from each sample were resuspended in 100 μl Stain Buffer (554656, BD Pharmingen) and incubated for 15 min on ice with 1 μg mouse IgG (ab37355, Abcam, 1:500) for Fc blocking. Then cells were incubated with primary monoclonal antibodies including rat anti-mouse Gr1/Ly-6G-Alexa Fluor 594 (Novus, 1:200), rat anti-mouse CD11b-FITC (R&D System,1:200) and rat anti-F4/80-APC (R&D System,1:200). Flow cytometry was performed using FACS LSR Fortessa X20 (BD Biosciences) and data were analyzed by BD FACSDiva 8.0.1 and FlowJo v10.8.1.