Eclipse e800 epifluorescence microscope

Alkaline comet assays were performed using a CometAssay kit (Trevigen) according to the manufacturer’s protocol. Splenic B cells were cultured for 48 hours in vitro and then treated with mock or 100 μM MMS treatment for 3 hours. Images were acquired by Nikon Eclipse E800 epifluorescence microscope and analyzed with OpenComet software.

LA4 cells were inoculated with RV and MHV-1 concurrently (RV+MHV) or sequentially with RV 6 h prior to inoculation with MHV-1 (RV/MHV), both viruses at a multiplicity of infection (MOI) of 1. Supernatant medium was collected every 6 h through the 24 h time point and at 48 h. Cells were removed by centrifugation, and MHV-1 titers in the medium were quantified by TCID₅₀ assay using 17Cl.1 cells. LA4 cells seeded onto coverslips were inoculated with RV and MHV-1 concurrently and fixed with formaldehyde 18 h post-infection. Viral antigens were fluorescently labeled in triton X-100-permeabilized cells using monoclonal antibody against MHV nucleocapsid protein (provided by Dr. Julian Leibowitz, Texas A&M University) and donkey anti-mouse-555 (Jackson Immuno Research), and RV1B antiserum (ATCC, V-113-501-558) and donkey anti-guinea pig-488 (Jackson Immuno Research). Nuclei were stained with DAPI and images were obtained using a Nikon Eclipse E800 epifluorescence microscope and Nikon DS-Ri1 camera.

As detailed previously, a single dose of purified WT or mutant NPC2 protein was added to the media of NPC2 mutant fibroblasts cultured on 8-well tissue culture slides (Nalgene), and allowed to incubate for 3 days. The final concentration of added protein was 0.4 nM. In keeping with prior literature (Ko et al., 2003 (link); Liou et al., 2006 (link); McCauliff et al., 2015 (link); Wang et al., 2010 (link)), since identical amounts of NPC2 protein were added to equivalent samples of cultured cells, we reasonably assume equivalent uptake of the various NPC2 proteins; notably all bind cholesterol similarly, implying that they fold normally and thus are of similar shape and size, precluding any potential difference in uptake via fluid phase endocytosis. Following incubations the cells were fixed and stained with 0.05 mg/mL filipin III (Fisher) and subsequently imaged on a Nikon Eclipse E800 epifluorescence microscope using a DAPI filter set. Filipin stain was quantified as a ratio of fluorescence intensity per unit cell area in treated and untreated conditions with the accompanying NIS-Elements software (Nikon Inc). Results are corrected for background fluorescence and are representative of an average of 80 to 100 cells per condition.