Plan apo objective lens

The HRWMs were imaged prior to perforation for overall structure and thickness and after perforation to inspect individual perforations. In preparation for imaging, the HRWMs were immersed in 1 mM Rhodamine B dissolved in PBS for 30 minutes, serially rinsed in PBS three times, and then soaked in PBS for 12 hours. Confocal imaging was performed using a Nikon A1R scanning confocal attachment on an Eclipse TiE microscope stand (Nikon Instruments, Melville, New York). Images of the entire HRWM were taken using a 10x/0.45 Plan Apo objective lens (Nikon) and required stitching (ImageJ-Fiji). Orthogonal slices were used to measure the thickness of the HRWM, with twenty-five measurements per HRWM, equally spaced in the orthogonal plane and the stacking direction (Fig. 2). To measure perforation sizes, images using a 20x/0.75 Plan Apo VC objective lens (Nikon) were projected in the stacking direction with maximum intensity to visualize a flattened membrane. Perforation sizes were measured manually and confirmed by scrolling through images slices in ImageJ-Fiji. If debris obstructed visualization of a perforation under fluorescence microscopy, transmitted white light was used instead. The areas of the perforations were measured by outlining each perforation and computing the area.

DNCCs or DTCCs were generated as described above and plated in eight chamber glass slides (BD Biosciences, San Jose, CA) at a concentration of 10⁵ cells/mL. Before lipid raft analysis, cells were first exposed to fluorescent lipids (NBD-PC or NBD-cholesterol; fluorescent in the green spectrum) at the indicated concentrations. Subsequently, lipid rafts were labeled by Vybrant^™ Alexa Fluor^™ 594 Lipid Raft Labeling Kit according to manufactures protocol (ThermoFisher Scientific). Briefly, cells were washed with PBS and CT-B (Cholera toxin subunit B) has been added with basal media (2 μg/mL) and incubated for 1 h at 4 °C. Cells were washed 3 times with cold PBS and anti CT-B (5 μL/mL in basal media) has been added to that. After incubation for 30 min at 4 °C, the cells were fixed with 4% paraformaldehyde for 20 min. Cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) and imaged by fluorescence or confocal microscopy. Fluorescent images were obtained using three channels on an NIKON Eclipse TI-U microscope with a 20×ELDW, 10 or 40× Plan-Apo objective lens (Nikon, Melville, NY). NIS Elements Viewer version 3.22 (Nikon, Melville, NY) software was used to capture the images to file.