Extended Data Fig. 1: Validation of virion yield and size measurements.
From: Influenza A virus rapidly adapts particle shape to environmental pressures
Unfractionated virus was purified by pelleting through a 20% sucrose cushion. a. Representative electron micrographs of five biologically independent samples. Samples include sucrose-cushion-purified virus (All), fractions obtained by 20–60% sucrose gradient centrifugation of the sucrose-cushion-purified virus (Spherical and Filamentous1-2), and a fraction obtained by centrifugal fractionation of the sucrose-cushion-purified virus (Filamentous3). b. Analysis of the 5 biologically independent samples in a, plus a buffer (Blank) using flow virometry. c. Plot of the percentage of filamentous virions within samples from a and b. Measurements were obtained either by flow virometry (gray bars), using the Filamentous Violet Side Scatter (VSSC) threshold indicated in b, or by identifying virions larger than 130nm by electron microscopy (white bars). d. Virion counts were determined for the samples from a and an additional nine fractions from the centrifugal fractionation experiments enriched either in filaments (Fil. Other) or spheres (Sph. Other) to various degrees. The counts were determined using flow virometry by either assuming a constant flow rate (black bars) or by accounting for flow rate fluctuations using a known concentration of fluorescent 170-nm beads (gray bars). Hemagglutination (HA) assays were performed on all samples. The HA units (HAU) and virion counts from the sucrose-cushion-purified virus were used to generate a scaling factor of 8.9 × 105 virions/HAU. The scaling factor we obtained is consistent with published literature that employed EM to correlate HAU to virion counts29,30,31. This factor was used to convert HAU values to estimated virion counts (red bars).
