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Normalization to Cell Number:

CellTag 700 Staining

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.

CellTag 700 staining

  • CellTag 700 Stain is a near-infrared fluorescent cell stain that provides accurate information to cell number for In-Cell Western™ applications
  • The stain accumulates in both the nucleus and the cytoplasm of permeabilized cells and provides linear fluorescent signal across a wide range of cell types and cell membranes (Fig. 1)
  • Fast and easy - staining is combined with secondary antibody incubation

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.

Cell Labeling with Reactive Dye

  • Covalently label cellular lysine residues on cellular proteins with
    IRDye® 800CW (Fig. 2 and 3) or IRDye 700DX reactive dyes1
  • Very inexpensive method with extreme sensitivity and wide linear range1 (~200 to 200,000 cells/well) (Fig. 3)
  • Results are unaffected by changes in nuclear DNA

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.

  • 1. Hoffman, GR et al. Assay Drug Dev Tech 8(2): 186-99 (2010)
  • IRDye 800CW Maleimide Labeling. Technical Note. LI-COR

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