Cell number normalization is a fast and inexpensive approach, because no additional antibodies are required. Options include CellTag 700 staining and cell labeling with reactive dye.
Figure 1. Linear Relationship between Fluorescence and Cell Number. Two-fold serial dilutions of A431 or NIH/3T3 cells were plated in 96-well plates. Cells were fixed, permeabilized, stained with CellTag 700 Stain (0.2 µM), and detected with Odyssey® Classic Infrared Imaging System. The Trim Signals were used to generate the graphs.
Figure 2. 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 3. 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.