Selective loss of chemokine receptor expression on leukocytes after cell isolation.

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CC...

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Autores principales:	Juan C Nieto, Elisabet Cantó, Carlos Zamora, M Angels Ortiz, Cándido Juárez, Silvia Vidal
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/03b6cb3fd27a47a48ccca6e5c1de174a
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spelling	oai:doaj.org-article:03b6cb3fd27a47a48ccca6e5c1de174a2021-11-18T07:26:11ZSelective loss of chemokine receptor expression on leukocytes after cell isolation.1932-620310.1371/journal.pone.0031297https://doaj.org/article/03b6cb3fd27a47a48ccca6e5c1de174a2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22403612/?tool=EBIhttps://doaj.org/toc/1932-6203Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5 ± 2.9%) whereas 32.8 ± 6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4 ± 7.5% and 57.1 ± 5.5%; Ficoll: 29.5 ± 2.2% and 5.4 ± 4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5 ± 0.4 and Ficoll: MFI 3.3 ± 0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2 ± 4.5% and Ficoll: 55 ± 4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.Juan C NietoElisabet CantóCarlos ZamoraM Angels OrtizCándido JuárezSilvia VidalPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 3, p e31297 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Juan C Nieto Elisabet Cantó Carlos Zamora M Angels Ortiz Cándido Juárez Silvia Vidal Selective loss of chemokine receptor expression on leukocytes after cell isolation.
description	Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5 ± 2.9%) whereas 32.8 ± 6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4 ± 7.5% and 57.1 ± 5.5%; Ficoll: 29.5 ± 2.2% and 5.4 ± 4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5 ± 0.4 and Ficoll: MFI 3.3 ± 0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2 ± 4.5% and Ficoll: 55 ± 4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.
format	article
author	Juan C Nieto Elisabet Cantó Carlos Zamora M Angels Ortiz Cándido Juárez Silvia Vidal
author_facet	Juan C Nieto Elisabet Cantó Carlos Zamora M Angels Ortiz Cándido Juárez Silvia Vidal
author_sort	Juan C Nieto
title	Selective loss of chemokine receptor expression on leukocytes after cell isolation.
title_short	Selective loss of chemokine receptor expression on leukocytes after cell isolation.
title_full	Selective loss of chemokine receptor expression on leukocytes after cell isolation.
title_fullStr	Selective loss of chemokine receptor expression on leukocytes after cell isolation.
title_full_unstemmed	Selective loss of chemokine receptor expression on leukocytes after cell isolation.
title_sort	selective loss of chemokine receptor expression on leukocytes after cell isolation.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/03b6cb3fd27a47a48ccca6e5c1de174a
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Selective loss of chemokine receptor expression on leukocytes after cell isolation.

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