Figure 1. Use of cell labeling for In-Cell Western normalization. A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format. Fixed cells were stained with phospho-rpS6 antibody and NHS ester reactive dye (for cell number). Dose dependent inhibition of phospho-rpS6-staining yielded an IC50 of 224 pM (n=4). B) Raw microplate image. Data courtesy of GR Hoffman.1
Figure 2. Linearity of cell labeling with IRDye 800CW reactive dye. Two-fold serial dilutions of HeLa or A431 cells were fixed and permeabilized. Cells were labeled with IRDye 800CW NHS ester at a 1:50,000 dilution. The method was linear from ~200-200,000 cells/well.