Microscope coverslip

Cells were cultured on microscope coverslips (Thermo Fisher Scientific, Rochester, NY, USA) and treated with gas (He+O₂) only, 2, or 4 kV of NTP, respectively. After 24 h, the slides were washed with PBS, fixed for 20 min in 3.7% formaldehyde, and re-hydrated in PBS. After blocking for 45 min in BSA (in 5% PBS), the slides were incubated for 1 h with polyclonal rabbit anti-p-FAK and-paxillin antibodies (1∶50; Cell Signaling Technology), washed with PBS, and incubated for 45 min with Alexa 488-labeled goat anti-rabbit antibodies (1∶250; Molecular Probes). After rinsing in PBS, Hoechst 33258 was added for 15 min to counterstain the nuclei. The slides were washed with PBS and mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA) then analyzed using fluorescence microscopy (Carl Zeiss).

Briefly, 1.5 × 10⁵ CHO-K1 cells (ATCC CCL-61) were seeded on a microscope coverslips (d = 18 mm, Fisherbrand) within 12-well plate with 1 ml complete medium (DMEM:F12 supplemented with 10% FBS). Cells were incubated under normal growth conditions (37 °C and 5% CO2) for overnight. CHO-K1 cells would reach about 80% confluency the next day, and they were cooled down on ice for 5 min first. Media were then removed and replaced with 400 μl cold buffers containing 12 μg cy5-labeled RBD protein and integrin primary antibody (7E2 Developmental Studies Hybridoma Bank) in HBSS with 5% BSA in the presence and absence of 1 mM manganese chloride, in the presence or absence of Cilengitide. Cells were incubated on ice for 1 h and incubated at 37 °C for another 30 min. After the incubation, cells were cooled down on ice for 5 min and then washed with cold HBSS, fixed with 4% formaldehyde for 20 min at room temperature. Cells were washed with HBSS and then incubated with the secondary antibody (Goat anti-mouse IgG H&L Alexa 488, Abcam, #ab150117). Cells were washed and sealed on microscope slides (25 × 75 mm, Fisherfinest) with Diamond Antifade mountant with DAPI. Cells were visualized in Cleveland Clinic Lerner Research Institute Light Microscopy core under widefield upright Microscope at 40x magnification using the LAS X software.

Mass photometry experiments were performed an OneMP instrument (Refeyn, Oxford, UK) at room temperature (43 (link), 44 (link)). The microscope coverslips (24 × 50 mm, Fisher Scientific) and precut 2 × 2 silicon gasket wells (Sigma) were cleaned with MilliQ water, isopropanol, and dried with clean nitrogen flow and assembled with an adjustable cover-slip rack. The instrument was calibrated using β-Amylase and Thyroglobulin from Refeyn as protein standards: 18 μL of the buffer was added in an empty well on the coverslip and the laser was focused; then 2 μL of β-Amylase or Thyroglobulin was added and mixed by pipetting; data acquisition was started immediately and the MP video was recorded using the AcquireMP software, and then the software DiscoverMP was used to analyze the data and generate the calibration function. To measure the mass of the protein of interest, 10 μL buffer was added to an empty well on the coverslip, and then 10 μL of Munc13-1 solution at various concentrations (from 8 nM to 170 nM) was added and well mixed. Data collection was then started and the MP video was recorded. DiscoverMP was used to process the data (45 (link)). The distribution of molecular mass was plotted as histograms and fit with Gaussian peaks to obtain the average mass of different species and determine the molecular weight of Munc13-1.