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A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells

Radziwon-Balicka, Aneta; Degn, Matilda; Johansson, Sara E. LU ; Warfvinge, Karin LU and Edvinsson, Lars LU (2017) In PLoS ONE 12(10).
Abstract

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... (More)

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.

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author
organization
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publication status
published
subject
in
PLoS ONE
volume
12
issue
10
publisher
Public Library of Science
external identifiers
  • scopus:85033409219
  • wos:000413910200007
ISSN
1932-6203
DOI
10.1371/journal.pone.0186504
language
English
LU publication?
yes
id
19ad9aa8-bab4-4099-bc37-7f3abd484ac7
date added to LUP
2017-11-21 07:56:32
date last changed
2018-01-16 13:26:04
@article{19ad9aa8-bab4-4099-bc37-7f3abd484ac7,
  abstract     = {<p>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 <sub>B</sub> receptor (ET<sub>B</sub>) 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 ET<sub>B</sub> 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 ET<sub>B</sub> 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 ET<sub>B</sub> receptors. The two subpopulation expressed the same level of ET<sub>B</sub> 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 ET<sub>B</sub> 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.</p>},
  articleno    = {e0186504},
  author       = {Radziwon-Balicka, Aneta and Degn, Matilda and Johansson, Sara E. and Warfvinge, Karin and Edvinsson, Lars},
  issn         = {1932-6203},
  language     = {eng},
  month        = {10},
  number       = {10},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells},
  url          = {http://dx.doi.org/10.1371/journal.pone.0186504},
  volume       = {12},
  year         = {2017},
}