Figure 1

In-Cell Western Optimization. (A) Tau levels from multiple cell lines were assessed by Western blot. H4 neuroglioma cells were chosen for the assay for several reasons including relatively robust tau expression, adhesion properties and human neural lineage. β-actin levels were assayed to control for variations in protein loading. (B) H4 cells were plated in a 96-well plate and tau immunoreactivity was determined by fluorescent (680 nm) secondary antibody detection using the Odyssey infrared scanner. Conditions were optimized to produce a low 5% well-to-well variation across the plate represented as CV (coefficient of variance) for rows B-G, and a signal-to-background ratio of >10-fold (12.9; background is empty wells in rows A and H). (C) Cells were plated at the densities indicated on the x-axis. Relative Fluorescence for triplicate wells were averaged and the standard deviation was calculated. Black diamonds (◆) indicate tau immunoreactivity with detection at 680 nm; gray boxes (■) indicate GAPDH immunoreactivity with detection at 800 nm