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

Nieto JC, Cantó E, Zamora C, Ortiz MA, Juárez C, Vidal S - PLoS ONE (2012)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institut Recerca Hospital de Sant Pau, Barcelona, Spain.

ABSTRACT
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.

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Effect of anticoagulant and the cell separation method on monocyte chemokine receptors expression.WB, Ficoll and Percoll isolated PBMC from healthy donors (n = 3) were collected in Vacutainer tubs containing sodium heparine or EDTA. Monocytes were CD14+ gated and CCR2 (clone 48607), CXCR3 and CXCR4 expression were determined. The results of a representative cytometric analysis were shown (n = 5) (isotype control □ , chemokine receptor ▪).
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pone-0031297-g002: Effect of anticoagulant and the cell separation method on monocyte chemokine receptors expression.WB, Ficoll and Percoll isolated PBMC from healthy donors (n = 3) were collected in Vacutainer tubs containing sodium heparine or EDTA. Monocytes were CD14+ gated and CCR2 (clone 48607), CXCR3 and CXCR4 expression were determined. The results of a representative cytometric analysis were shown (n = 5) (isotype control □ , chemokine receptor ▪).

Mentions: To evaluate the influence of anticoagulants, we compared the influence of heparin and Ca2+ chelators such as EDTA on the chemokine receptor expression of Ficoll-isolated monocytes. Monocytes collected in heparin- or EDTA- WB expressed similar levels of CCR2 and CXCR3. A significant reduction of the CXCR4 expression was observed on the EDTA-WB monocytes (MFI 5.43±0.35 in heparin WB and MFI 1.2±0.04 in EDTA WB p<0.05). The use of EDTA instead of heparin did not prevent CCR2 and CXCR3 downregulation after Ficoll isolation (CCR2: MFI 3.8±0.8 in heparin WB, 3.0±0.07 in EDTA WB, Ficoll MFI 1.6±0.6 in heparin, MFI 1.0±0.07 in EDTA. CXCR3: MFI 1.3±0.7 in heparin WB, 1.4±0.3 in EDTA WB, Ficoll MFI 0.3±0.6 in heparin, MFI 0.5±0.01 in EDTA). The downregulation of chemokine receptors on the Ficoll isolated monocytes was not influenced by the type of anticoagulant into which the peripheral blood was collected (Fig. 2).


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

Nieto JC, Cantó E, Zamora C, Ortiz MA, Juárez C, Vidal S - PLoS ONE (2012)

Effect of anticoagulant and the cell separation method on monocyte chemokine receptors expression.WB, Ficoll and Percoll isolated PBMC from healthy donors (n = 3) were collected in Vacutainer tubs containing sodium heparine or EDTA. Monocytes were CD14+ gated and CCR2 (clone 48607), CXCR3 and CXCR4 expression were determined. The results of a representative cytometric analysis were shown (n = 5) (isotype control □ , chemokine receptor ▪).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293867&req=5

pone-0031297-g002: Effect of anticoagulant and the cell separation method on monocyte chemokine receptors expression.WB, Ficoll and Percoll isolated PBMC from healthy donors (n = 3) were collected in Vacutainer tubs containing sodium heparine or EDTA. Monocytes were CD14+ gated and CCR2 (clone 48607), CXCR3 and CXCR4 expression were determined. The results of a representative cytometric analysis were shown (n = 5) (isotype control □ , chemokine receptor ▪).
Mentions: To evaluate the influence of anticoagulants, we compared the influence of heparin and Ca2+ chelators such as EDTA on the chemokine receptor expression of Ficoll-isolated monocytes. Monocytes collected in heparin- or EDTA- WB expressed similar levels of CCR2 and CXCR3. A significant reduction of the CXCR4 expression was observed on the EDTA-WB monocytes (MFI 5.43±0.35 in heparin WB and MFI 1.2±0.04 in EDTA WB p<0.05). The use of EDTA instead of heparin did not prevent CCR2 and CXCR3 downregulation after Ficoll isolation (CCR2: MFI 3.8±0.8 in heparin WB, 3.0±0.07 in EDTA WB, Ficoll MFI 1.6±0.6 in heparin, MFI 1.0±0.07 in EDTA. CXCR3: MFI 1.3±0.7 in heparin WB, 1.4±0.3 in EDTA WB, Ficoll MFI 0.3±0.6 in heparin, MFI 0.5±0.01 in EDTA). The downregulation of chemokine receptors on the Ficoll isolated monocytes was not influenced by the type of anticoagulant into which the peripheral blood was collected (Fig. 2).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institut Recerca Hospital de Sant Pau, Barcelona, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus