Cellometer

Recombinant VSV expressing eGFP and the Lassa virus glycoprotein (rVSV-ΔG-LASV) was prepared as previously described^{11 (link),70 (link)}. Following enzymatic cell-surface display in 96-well plate format, cells were gently washed with 100 µL DPBS three times. For each infection experiment, cells from three wells were harvested using 1X trypsin-EDTA and counted using a Nexcelom Cellometer to determine the average cell number per well to determine multiplicity of infection (MOI) calculations. Cells were infected with rVSV-ΔG-LASV at an MOI of 1 (1 virion per cell) in 50 µL of cell culture media for 1 h at 37 °C. Cells were then gently washed with 100 µL DPBS three times, and 100 µL of complete cell culture media was applied to each well. Expression of eGFP was analyzed 24 h post-infection by fluorescence microscopy using a fluorescence microscope (Nikon Eclipse, TE2000-S) and captured using a Qimaging (Retiga 1300i Fast) camera and Qcapture version 2.90.1 software, followed by harvesting of cells and quantification of the number of eGFP-positive cells relative to the total number of cells using a Nexcelom Cellometer. All experiments were performed at technical triplicate or greater. Raw data values were analyzed and plotted using GraphPad Prism 9.

Using the acridine orange (AO) and propidium iodide (PI) staining, viability was determined in U937 and MM6 cells for plating and after treatment with flavoring chemicals and e-liquids. AO/PI staining and viability determination was performed in 20 μL of cells combined with 20 μL of AO/PI staining solution. Finally, 20 μL of stained cells were then added to a Cellometer counting chamber and analyzed using a fluorescent Cellometer (Nexcelom Bioscience, Lawrence MA). At the end of the analysis, the Cellometer automatically reported live and dead cell concentration as a percentage.