Dataset Information

Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain.

ABSTRACT:

Introduction

Fibrocytes are emerging myeloid-derived circulating cells that can migrate into damaged tissues and usually contribute to their repair. Key features of fibrocytes include the expression myeloid markers, production of extracellular matrix proteins, and secretion of various humoral factors that activate resident fibroblasts; they also have the potential to differentiate into fibroblasts. However, no specific surface markers have been identified to identify fibrocytes in vivo. One reason could be that the method used to detect fibrocytes requires intracellular collagen staining.

Methods

In the present study, to establish an improved method for the detection of lung fibrocytes and to analyze viable fibrocytes, we used collagen I(?)2-green fluorescent protein (Col-GFP) reporter mice, which had undergone the intratracheal instillation of bleomycin (BLM).

Results

Using flow cytometry to gate out cells with autofluorescence, we clearly found that CD45⁺ GFP⁺ cells resided in the lungs of Col-GFP mice at a steady state and these cells increased after BLM injury, peaking at Day 14. These cells expressed not only known cell surface markers of fibrocytes, but also some novel markers, in addition to a low level of collagen I in comparison to CD45^- GFP⁺ cells.

Conclusion

Our findings suggest that the improved method can be a useful for the detection of pure lung fibrocytes and allows us to further analyze the characteristics of viable fibrocytes.

SUBMITTER: Kawano H

PROVIDER: S-EPMC7860606 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Publications

Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain.

Kawano Hiroshi H Koyama Kazuya K Nishimura Haruka H Toyoda Yuko Y Kagawa Kozo K Sato Seidai S Naito Nobuhito N Goto Hisatsugu H Inagaki Yutaka Y Nishioka Yasuhiko Y

Immunity, inflammation and disease 20201224 1

<h4>Introduction</h4>Fibrocytes are emerging myeloid-derived circulating cells that can migrate into damaged tissues and usually contribute to their repair. Key features of fibrocytes include the expression myeloid markers, production of extracellular matrix proteins, and secretion of various humoral factors that activate resident fibroblasts; they also have the potential to differentiate into fibroblasts. However, no specific surface markers have been identified to identify fibrocytes in vivo. ...[more]

PMID: 33369271

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Project description:Despite the remarkable evolution of flow cytometers, fluorescent probes, and flow cytometry analysis software, most users still follow the same ways for data analysis. Conventional flow cytometry analysis relies on the creation of dot plot sequences, based on two fluorescence parameters at a time, to evidence phenotypically distinct populations. Thus, reaching conclusions about the biological characteristics of the samples is a fragmented and challenging process. We present here the MCTA (Multiparametric Color Tendency Analysis), a method for data analysis that considers multiple labelings simultaneously, extending and complementing conventional analysis. The MCTA method executes the background fluorescence exclusion, spillover compensation, and a user-defined gating strategy for subpopulation analysis. The results are then presented in conventional FSC x SSC dot plots with statistical data. For each event, the method converts each of the multiple fluorescence colors under analysis into a vector, with longer vectors being attributed to more intense labelings. Then, the MCTA generates a resultant vector, which is therefore mostly influenced by predominant labelings. The radial position of this resultant vector corresponds to a resultant color, making it easy to visualize phenotypic modulations among cellular subpopulations. Besides, it is a deterministic method that quickly assigns a resulting color to all events that obey the gating strategy, with no polymeric regions defined by the user or downsampling. The MCTA application generates a single dot plot showing all events in the FCS file, but a resultant color is attributed only to those that obey the gating strategy. Therefore, it can also help to evidence rare events or unpredicted subpopulations naturally excluded from the regions defined by the user. We believe that the MCTA method adds a new perspective over multiparametric flow cytometry analysis while evidencing modulations of molecular labeling profiles based on multiple fluorescences. Availability and implementation: The instructions for the MCTA application is freely available at https://github.com/flowcytometry/MCTA.

Dataset Information

Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain.

Introduction

Methods

Results

Conclusion

Publications

Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain.

Similar Datasets

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