Overview -- All Autophagy Positive Aspects As well as Disadvantages

All viral particles and polystyrene beads were counted in at least 10 randomly chosen squares on each grid. The volume was calculated by dividing the number of beads counted by the known concentration per mL (2E+10). The bead solution: viral stock solution ratio was 1:1, so VP/mL was determined by dividing counted particles by the calculated volume of sample: V=beads?countedbead?concentration?per?ml (1) Autophagy Viral particles per mL=viral?particles?countedV (2) Final virus particle concentration of the seed stock was established using results from samples with countable number of particles on all three grids and factoring in the dilution of the sample. 2.4.2. ViroCyt? Virus Counter 2100 Instrument The VC reagent kit (ViroCyt) was used following the manufacturer-recommended procedures. The instrument performance was validated prior to testing samples by running a non-biological positive control (stained beads performance validation Etoposide standard used as an instrument performance check) and cleanliness control to verify the flow path was clean. Briefly, 300 ��L of each sample was stained using 150 ��L of Combo Dye solution, incubated in the dark at room temperature for 30 min, and analyzed in the VC. Samples were tested in triplicate with inter-sample washes and a cleanliness control run between each sample. Results were automatically analyzed by the instrument software and reported as VP/mL. A negative control sample was diluted and tested to establish the sample quantification limit [13]. The negative control sample was a clarified medium collected from uninfected Vero E-6 cells, prepared in parallel with our EBOV preparation. All VC results greater than this value were considered statistically distinguishable from the negative control (background) and therefore reported. The sample quantification limit was determined using the following equation: Sample quantification limit=Xneg+t99%(��neg) (3) Xneg is the mean value of the negative control samples; ��neg is the standard deviation of the negative control samples; and t99% is the t value for N-1 degrees of freedom at the 99% confidence level. Final virus particle concentration of the seed stock was established using all samples whose VP/mL counts were above the sample quantification limit and within the linear range of the instrument and factoring in the dilution of the sample. 2.5. Statistical Analysis Microsoft Excel MEK inhibition 2007 (Redmond, WA, USA) was used for linear-regression analysis. GraphPad Prism v5 software (LaJolla, CA, USA) was used to perform Pearson correlation analysis to determine the correlation between each assay (correlation coefficients (r) and p-values). A p-value of