Vybrant dio cell labeling solution

Monocyte-derived macrophages were first stained with 2.5 µg/ml of Vybrant DiO Cell-Labeling Solution (Thermo Scientific) diluted in PBS. The cells were then washed three times with PBS and incubated with eFluor 670 spermatozoa in the absence or presence of SEM fibrils using conditions described above. After 3 hr, macrophages were washed 3x with PBS, trypsinized, and stored in 1% PFA at 4°C in the dark until imaging. Confocal imaging was carried out using a Nikon Eclipse Ti-E inverted microscope equipped with a Yokogawa CSU22 spinning disk confocal scanner, a Sutter emission Lambda filter wheel adapter with ET460/50m, ET525/50m, ET645/65m and ET700/75m filters, a Prior motorized stage with Piezo Z-drive, and a Photometrics Evolve 512 Delta EMCCD Camera. Images were acquired with Micro-Manager software version 1.4.21. Image scanning was executed employing a Plan Apo VC 100x/1.4 Oil (DIC N2/100X I) objective using 488 nm and 640 nm, 100 mW Coherent OBIS lasers. The Piezo Z-drive was set for fast acquisition and z-steps at 0.40 µm. Image analysis was performed using ImageJ software version 1.48 with the Deconvolution lab (EPFL) and 3D viewer (B. Schmid) plugins installed. All acquisition and analysis parameters were maintained constant for all samples.

Acti-stain
488 phalloidin was purchased from Cytoskeleton Inc. Advanced Dulbecco’s
modified Eagle’s medium (aDMEM), Dulbecco’s modified
Eagle’s medium–Nutrient Mixture F-12 l-glutamine
(DMEM/F12), Dulbecco’s phosphate-buffered saline (DPBS) 10×,
glutamine, horse serum, penicillin–streptomycin, phosphate-buffered
saline (PBS), propidium iodide (PI), and Vybrant DiO cell-labeling
solution were acquired from Thermo Fisher Scientific. Anti-Rabbit
IgG (H+L), CF 647 antibody produced in goat (SAB4600184), bovine serum
albumin (BSA), calcein AM, cholera toxin, doxorubicin, epidermal growth
factor (EGF), fetal bovine serum (FBS), hydrocortisone, insulin from
bovine pancreas, paraformaldehyde (PFA), Triton X-100, and 4′,6-diamidino-2-phenylindole
(DAPI) were purchased from Sigma-Aldrich. Anti-collagen I antibody
(ab34710), anti-collagen III antibody (ab7778), anti-collagen IV antibody
(ab6586), anti-fibronectin antibody (ab2413), and goat serum were
purchased from Abcam. Collagen type I (Col1) was isolated from rat
tail tendons.^{68 (link)} The Col1 content was measured
by microBCA (Thermo Fisher Scientific) as previously described.^{56 (link)}

For TIRFM, AECs were grown on fibronectin-coated μ-slide 8 well glass bottom slides (Ibidi) for 4 days and stained with 5 μl/ml Vybrant DiO cell labeling solution (Thermofisher) for 10 min at 37 °C. Life imaging was performed in the imaging facility of the Molecular Biophysics group, HU Berlin on a Confocal Laser Scanning Microscope (Olympus FV-1000MPE) equipped with a TIRFM upgrade at 37 °C and 5% CO₂ using a 63x oil objective and a cooled CCD camera. Images were taken at indicated time points and analysed using FijI. Particle analysis was performed from binary images after thresholding (method Isodata). Total intensity of attachment from all detected particles was measured.