Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis
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 highe...
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Published in | Experimental eye research Vol. 93; no. 6; pp. 956 - 962 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
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Elsevier Ltd
01.12.2011
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Abstract | 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. |
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AbstractList | 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. 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. |
Author | Martino, Mario Garcia-Gutierrez, Maria T. Pastor, J. Carlos Singh, Amar K. Fernandez-Bueno, Ivan Hileeto, Denise Reinoso, Roberto Srivastava, Girish K. Alonso, Nieves Fernández Pigazo Merino, Jose M. Corell, Alfredo |
Author_xml | – sequence: 1 givenname: Girish K. surname: Srivastava fullname: Srivastava, Girish K. email: girish@ioba.med.uva.es organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 2 givenname: Roberto surname: Reinoso fullname: Reinoso, Roberto organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 3 givenname: Amar K. surname: Singh fullname: Singh, Amar K. organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 4 givenname: Ivan surname: Fernandez-Bueno fullname: Fernandez-Bueno, Ivan organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 5 givenname: Denise surname: Hileeto fullname: Hileeto, Denise organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 6 givenname: Mario surname: Martino fullname: Martino, Mario organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 7 givenname: Maria T. surname: Garcia-Gutierrez fullname: Garcia-Gutierrez, Maria T. organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 8 givenname: Jose M. surname: Pigazo Merino fullname: Pigazo Merino, Jose M. organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 9 givenname: Nieves Fernández surname: Alonso fullname: Alonso, Nieves Fernández organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 10 givenname: Alfredo surname: Corell fullname: Corell, Alfredo organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain – sequence: 11 givenname: J. Carlos surname: Pastor fullname: Pastor, J. Carlos organization: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain |
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Keywords | autofluorescence immunohistochemistry (IHC) flow cytometry (FC) retinal pigment epithelial (RPE) cells trypan blue |
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Snippet | Retinal pigment epithelial (RPE) cells are currently in the “spotlight” of cell therapy approaches to some retinal diseases. The analysis of the expressed... Retinal pigment epithelial (RPE) cells are currently in the "spotlight" of cell therapy approaches to some retinal diseases. The analysis of the expressed... |
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SubjectTerms | 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 |
Title | Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis |
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