A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells.
There is a need to develop new techniques for quantitative measurement of receptors expression on particular vasculature cells types. Here, we describe and demonstrate a novel method to measure quantitatively and simultaneously the expression of endothelin B receptor (ETB) on vascular smooth muscle...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2017-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5662092?pdf=render |
| _version_ | 1819264254983798784 |
|---|---|
| author | Aneta Radziwon-Balicka Matilda Degn Sara E Johansson Karin Warfvinge Lars Edvinsson |
| author_facet | Aneta Radziwon-Balicka Matilda Degn Sara E Johansson Karin Warfvinge Lars Edvinsson |
| author_sort | Aneta Radziwon-Balicka |
| collection | DOAJ |
| description | There is a need to develop new techniques for quantitative measurement of receptors expression on particular vasculature cells types. Here, we describe and demonstrate a novel method to measure quantitatively and simultaneously the expression of endothelin B receptor (ETB) on vascular smooth muscle cells (VSMC). We isolated cells from male rat tissues such as: brain pial, brain intraparenchymal and retina vessels. To analyze solid tissues, a single-cell suspension was prepared by a combined mechanic and enzymatic process. The cells were stained with Fixable Viability Dye, followed by fixation, permeabilization and antibodies staining. The expression of ETB receptors on VSMC was measured by flow-cytometry and visualized by fluorescence microscopy. We obtained a high percentage of viable cells 87.6% ± 1.5% pial; 84.6% ± 4.3% parenchymal and 90.6% ± 4% retina after isolation of single cells. We performed a quantitative measurement of ETB receptor expression on VSMC and we identified two subpopulations of VSMC based on their expression of smooth muscle cells marker SM22α. The results obtained from pial vessels are statistically significant (38.4% ± 4% vs 9.8% ± 3.32%) between the two subpopulations of VSMC. The results obtained from intraparenchymal and retina vessels were not statistically significant. By specific gating on two subpopulations, we were able to quantify the expression of ETB receptors. The two subpopulation expressed the same level of ETB receptor (p = 0.45; p = 0.3; p = 0.42) in pial, parenchymal and retina vessels, respectively. We applied our method to the animals after induction of subarachnoid hemorrhage (SAH). There was statistically significant expression of ETB receptor (p = 0.02) on VSMC between sham 61.4% ± 4% and SAH 77.4% ± 4% rats pial vessels. The presented technique is able to quantitatively and selectively measure the level of protein expression on VSMC. The entire technique is optimized for rat tissue; however the protocol can also be adapted for other species. |
| first_indexed | 2024-12-23T20:26:34Z |
| format | Article |
| id | doaj.art-9fc8fc3a81fa43b2be2b6d5b7585f758 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-23T20:26:34Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-9fc8fc3a81fa43b2be2b6d5b7585f7582022-12-21T17:32:22ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011210e018650410.1371/journal.pone.0186504A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells.Aneta Radziwon-BalickaMatilda DegnSara E JohanssonKarin WarfvingeLars EdvinssonThere is a need to develop new techniques for quantitative measurement of receptors expression on particular vasculature cells types. Here, we describe and demonstrate a novel method to measure quantitatively and simultaneously the expression of endothelin B receptor (ETB) on vascular smooth muscle cells (VSMC). We isolated cells from male rat tissues such as: brain pial, brain intraparenchymal and retina vessels. To analyze solid tissues, a single-cell suspension was prepared by a combined mechanic and enzymatic process. The cells were stained with Fixable Viability Dye, followed by fixation, permeabilization and antibodies staining. The expression of ETB receptors on VSMC was measured by flow-cytometry and visualized by fluorescence microscopy. We obtained a high percentage of viable cells 87.6% ± 1.5% pial; 84.6% ± 4.3% parenchymal and 90.6% ± 4% retina after isolation of single cells. We performed a quantitative measurement of ETB receptor expression on VSMC and we identified two subpopulations of VSMC based on their expression of smooth muscle cells marker SM22α. The results obtained from pial vessels are statistically significant (38.4% ± 4% vs 9.8% ± 3.32%) between the two subpopulations of VSMC. The results obtained from intraparenchymal and retina vessels were not statistically significant. By specific gating on two subpopulations, we were able to quantify the expression of ETB receptors. The two subpopulation expressed the same level of ETB receptor (p = 0.45; p = 0.3; p = 0.42) in pial, parenchymal and retina vessels, respectively. We applied our method to the animals after induction of subarachnoid hemorrhage (SAH). There was statistically significant expression of ETB receptor (p = 0.02) on VSMC between sham 61.4% ± 4% and SAH 77.4% ± 4% rats pial vessels. The presented technique is able to quantitatively and selectively measure the level of protein expression on VSMC. The entire technique is optimized for rat tissue; however the protocol can also be adapted for other species.http://europepmc.org/articles/PMC5662092?pdf=render |
| spellingShingle | Aneta Radziwon-Balicka Matilda Degn Sara E Johansson Karin Warfvinge Lars Edvinsson A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells. PLoS ONE |
| title | A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells. |
| title_full | A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells. |
| title_fullStr | A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells. |
| title_full_unstemmed | A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells. |
| title_short | A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells. |
| title_sort | novel multicolor flow cytometry application for quantitative detection of receptors on vascular smooth muscle cells |
| url | http://europepmc.org/articles/PMC5662092?pdf=render |
| work_keys_str_mv | AT anetaradziwonbalicka anovelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT matildadegn anovelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT saraejohansson anovelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT karinwarfvinge anovelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT larsedvinsson anovelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT anetaradziwonbalicka novelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT matildadegn novelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT saraejohansson novelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT karinwarfvinge novelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells AT larsedvinsson novelmulticolorflowcytometryapplicationforquantitativedetectionofreceptorsonvascularsmoothmusclecells |