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Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

Zhao Y, Zhao H, Zhang Y, Tsatralis T, Cao Q, Wang Y, Wang Y, Wang YM, Alexander SI, Harris DC, Zheng G - BMC Cell Biol. (2014)

Bottom Line: The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%.Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.

ABSTRACT

Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.

Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.

Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

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Immunofluorescence staining assessment of MicroBeads isolated peritubular endothelial cells after 2 days culture. Immunofluorescence staining of isolated peritubular endothelial cells against VE-cadherin (green, A), FSP-1 (green, indicated by white arrow, B), CD11c (green, C), F4/80 (green, E), and positive control for CD11c (primary spleen dendritic cells, green, D) and F4/80 (primary spleen macrophage, green, F), Immunofluorescence staining against pericyte marker CD73 (green, G) and PDGFR-β (green, I), and positive control for CD73 (frozen kidney sections of BALB/c mouse, green, H) and PDGFR-β (frozen kidney sections of BALB/c mouse, green, J). (K) Statistical analysis of each individual marker immunofluorescent stained and quantified as a percentage of positively stained cells against total cells (2000). Data are expressed as mean ± SEM with N = 10 for each experimental group. Original magnification was x 200; original magnification for pericytes staining was x 400. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).
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Fig3: Immunofluorescence staining assessment of MicroBeads isolated peritubular endothelial cells after 2 days culture. Immunofluorescence staining of isolated peritubular endothelial cells against VE-cadherin (green, A), FSP-1 (green, indicated by white arrow, B), CD11c (green, C), F4/80 (green, E), and positive control for CD11c (primary spleen dendritic cells, green, D) and F4/80 (primary spleen macrophage, green, F), Immunofluorescence staining against pericyte marker CD73 (green, G) and PDGFR-β (green, I), and positive control for CD73 (frozen kidney sections of BALB/c mouse, green, H) and PDGFR-β (frozen kidney sections of BALB/c mouse, green, J). (K) Statistical analysis of each individual marker immunofluorescent stained and quantified as a percentage of positively stained cells against total cells (2000). Data are expressed as mean ± SEM with N = 10 for each experimental group. Original magnification was x 200; original magnification for pericytes staining was x 400. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).

Mentions: The high purity of isolated MRPECs shown by FACS analysis was further confirmed with immunofluorescent staining. After two days of culture, immunofluorescent staining of isolated MRPECs showed that all cells were VE-cadherin positive and few cells were FSP-1 positive (Figure 3A, B). Immunofluorescent staining for CD11c and F4/80 also suggested that no dendritic or macrophage cells were present (Figure 3C, E). To confirm the absence of pericyte contamination, cells were stained for CD73 and PDGFR-β. Results showed that almost none of the cells expressed CD73 or PDGFR-β (Figure 3G, I). Therefore, taken together the FACS analysis and immunofluorescent results showed that MRPECs of high purity were successfully isolated from mouse renal tissue.Figure 3


Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

Zhao Y, Zhao H, Zhang Y, Tsatralis T, Cao Q, Wang Y, Wang Y, Wang YM, Alexander SI, Harris DC, Zheng G - BMC Cell Biol. (2014)

Immunofluorescence staining assessment of MicroBeads isolated peritubular endothelial cells after 2 days culture. Immunofluorescence staining of isolated peritubular endothelial cells against VE-cadherin (green, A), FSP-1 (green, indicated by white arrow, B), CD11c (green, C), F4/80 (green, E), and positive control for CD11c (primary spleen dendritic cells, green, D) and F4/80 (primary spleen macrophage, green, F), Immunofluorescence staining against pericyte marker CD73 (green, G) and PDGFR-β (green, I), and positive control for CD73 (frozen kidney sections of BALB/c mouse, green, H) and PDGFR-β (frozen kidney sections of BALB/c mouse, green, J). (K) Statistical analysis of each individual marker immunofluorescent stained and quantified as a percentage of positively stained cells against total cells (2000). Data are expressed as mean ± SEM with N = 10 for each experimental group. Original magnification was x 200; original magnification for pericytes staining was x 400. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260259&req=5

Fig3: Immunofluorescence staining assessment of MicroBeads isolated peritubular endothelial cells after 2 days culture. Immunofluorescence staining of isolated peritubular endothelial cells against VE-cadherin (green, A), FSP-1 (green, indicated by white arrow, B), CD11c (green, C), F4/80 (green, E), and positive control for CD11c (primary spleen dendritic cells, green, D) and F4/80 (primary spleen macrophage, green, F), Immunofluorescence staining against pericyte marker CD73 (green, G) and PDGFR-β (green, I), and positive control for CD73 (frozen kidney sections of BALB/c mouse, green, H) and PDGFR-β (frozen kidney sections of BALB/c mouse, green, J). (K) Statistical analysis of each individual marker immunofluorescent stained and quantified as a percentage of positively stained cells against total cells (2000). Data are expressed as mean ± SEM with N = 10 for each experimental group. Original magnification was x 200; original magnification for pericytes staining was x 400. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).
Mentions: The high purity of isolated MRPECs shown by FACS analysis was further confirmed with immunofluorescent staining. After two days of culture, immunofluorescent staining of isolated MRPECs showed that all cells were VE-cadherin positive and few cells were FSP-1 positive (Figure 3A, B). Immunofluorescent staining for CD11c and F4/80 also suggested that no dendritic or macrophage cells were present (Figure 3C, E). To confirm the absence of pericyte contamination, cells were stained for CD73 and PDGFR-β. Results showed that almost none of the cells expressed CD73 or PDGFR-β (Figure 3G, I). Therefore, taken together the FACS analysis and immunofluorescent results showed that MRPECs of high purity were successfully isolated from mouse renal tissue.Figure 3

Bottom Line: The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%.Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.

ABSTRACT

Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.

Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.

Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

Show MeSH
Related in: MedlinePlus