Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis

• Published in: Experimental eye research Vol. 93; no. 6; pp. 956 - 962
• Main Authors: Srivastava, Girish K., Reinoso, Roberto, Singh, Amar K., Fernandez-Bueno, Ivan, Hileeto, Denise, Martino, Mario, Garcia-Gutierrez, Maria T., Pigazo Merino, Jose M., Alonso, Nieves Fernández, Corell, Alfredo, Pastor, J. Carlos
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2011
• Subjects: Animals; autofluorescence; Cells, Cultured; Coloring Agents; Epithelial Cells - metabolism; Eye Proteins - metabolism; Flow Cytometry; flow cytometry (FC); Fluorescent Antibody Technique; immunohistochemistry (IHC); Paraffin Embedding; Reproducibility of Results; retinal pigment epithelial (RPE) cells; Retinal Pigment Epithelium - metabolism; Staining and Labeling - methods; Swine; Trypan Blue; autofluorescence; immunohistochemistry (IHC); flow cytometry (FC); retinal pigment epithelial (RPE) cells; trypan blue
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2011.07.002

Retinal pigment epithelial (RPE) cells are currently in the “spotlight” of cell therapy approaches to some retinal diseases. The analysis of the expressed proteins of RPE primary cells is an essential step for many of these approaches. But the emission of autofluorescence by RPE cells produces higher background noise interference thereby creating an impediment to this analysis. Trypan Blue (TB), a routinely used counterstain, has the capacity to quench this autofluorescence, if it is used in optimized concentration. The results from the method developed in our study indicate that incubation of the cultured RPE cells with 20 μg/ml of TB after immunolabelling (post-treatment) as well as incubation of the retinal tissue specimens with same concentration before paraffin embedding, sectioning and immunolabelling (pre-treatment) can be applied to effectively quench the autofluorescence of RPE cells. Thus it can facilitate the evaluation of expressed cellular proteins in experimental as well as in pathological conditions, fulfilling the current requirement for developing a method which can serve to eliminate the autofluorescence of the cells, not only in cell cultures but also in tissues samples. This method should significantly increase the quality and value of RPE cell protein analysis, as well as other cell protein analysis performed by Flow cytometry (FC) and Immunohistochemistry (IHC) techniques. ► Trypan Blue to Quench Autofluorescence for Improving RPE cell Protein Analysis. ► RPE cells incubation with 20 μg/ml of Trypan Blue quenches cell Autofluorescence. ► FC cell analysis requires post-treatment of RPE cells with Trypan Blue. ► IHC cell analysis requires pre-treatment of RPE cells with Trypan Blue. ► Significant increase in quality and value of cell analysis using methods developed.