Non-classical monocytes promote neurovascular repair in cerebral small vessel disease associated with microinfarctions via CX3CR1

Lecordier, Sarah; Menet, Romain; Allain, Anne-Sophie; ElAli, Ayman

doi:10.1177/0271678x231183742

Cited by 2 publications

(2 citation statements)

References 76 publications

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“…Monocytes from the peripheral blood and spleen of C57Bl/6 mice show no inverse correlation of Ly6C and CX3CR1 expression by ow cytometry In a rst step, we aimed to set up a ow cytometry panel to characterise classical and non-classical monocytes based on Ly6C and CX3CR1 as suggested in the literature [1,[24][25][26] . Thus, we isolated cells from the peripheral blood of C57Bl/6 mice and analysed them by ow cytometry.…”

Section: Resultsmentioning

confidence: 99%

Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

Sommer,

Garibagaoglu,

Wiendl

et al. 2023

Preprint

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Monocytes as well as downstream macrophages and dendritic cells are essential players of the immune system fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies in reporter models, mouse monocytes are divided into a classical and a non-classical subset based on their inversely correlating surface expression of Ly6C and CX3CR1. Here, we analysed the expression of CX3CR1 by flow cytometry using several validated fluorochrome-coupled CX3CR1 antibodies and compared them with the reporter gene signal of a Cx3cr1GFP reporter mouse strain as well as of tamoxifen-inducible Cx3cr1 reporter mice. Although we were able to validate the specificity of several fluorochrome-coupled CX3CR1 flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in peripheral blood and spleen, when stained with these antibodies. To the contrary, in reporter mice, we were able to reproduce the inverse correlation of CX3CR1 reporter gene signal and Ly6C surface expression. As determined by qPCR, the Cx3cr1 mRNA expression correlated with the GFP-reporter gene expression as quantified by flow cytometry. In conclusion, our data suggest that there is differential transcription, but not surface expression of CX3CR1 between classical and non-classical monocytes, which limits the suitability of CX3CR1 for phenotyping monocyte subsets by antibody staining.

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Section: Resultsmentioning

confidence: 99%

Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

Sommer,

Garibagaoglu,

Wiendl

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…As the first step, we aimed to set up a flow cytometry panel to characterize classical and non-classical monocytes based on Ly6C and CX3CR1, as suggested in the literature [1,[36][37][38]. Thus, we isolated cells from the peripheral blood of C57Bl/6 mice and analyzed them by flow cytometry.…”

Section: Monocytes From the Peripheral Blood And Spleen Of C57bl/6 Mi...mentioning

confidence: 99%

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies

Sommer,

Garibagaoglu,

Paap

et al. 2024

Cells

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Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

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Non-classical monocytes promote neurovascular repair in cerebral small vessel disease associated with microinfarctions via CX3CR1

Cited by 2 publications

References 76 publications

Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies

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