Dual function of PDGFRalpha positive progenitor cells during regeneration and revascularization stimuli

Published: 30-12-2019| Version 2 | DOI: 10.17632/wkg6844nwv.2
Contributor:
maria paola santini

Description

These data show that PDGFRa+ cells are stromal cells and promote increased vessel stability after hind limb ischemia. On the contrary, differentiated cells increased vessel permeability. The overall analysis described a dual function of PDGFRa+ cells in tissue revascularization. Folder Flow cytometry and sorting of PDGFRα+GFP+ cells: In this folder there is an example of flow cytometry for sorting PDGFRα+ cells (n=3). Adductor skeletal muscles were isolated from 3 month-old male WT control mice and left un-labeled (also without DAPI) to set gates for sorting, or labeled with anti-PDGFRα APC-conjugated antibody and DAPI to sort for GFP+PDGFRα+ co-positive cells from PdgfrαH2B-eGfp mice. The Hoechst Blue channel distinguishes DAPI(-) live cells from dead (DAPI+) cells. Taking the live DAPI(-) population, FITC was used to specify for PdgfrαH2B-eGfp+ cells. In the final panel, the FITC (x axis) and APC (y axis) specifies for cells double positive for GFP and APC (after labeling with anti-PDGFRα APC-conjugated antibody). Sorted live GFP+PDGFRα+ co-positive cells were processed for further analyses (RNAseq and adoptive transfer experiments). Folder evans blue dye to measure vessel permeability: Vessel permeability was assessed as described previously (Radu and Chernoff, 2013). Briefly, 200 μL of 1% Evans blue dye (Sigma Aldrich, Inc.) diluted in 1x PBS was injected via tail vein. The adductor muscle was isolated 30 minutes after injection and weighed. Dissected tissues were left for 36 hours in 500 μL of formamide at 55°C prior to spectrophotometric measurements (Abs=610nm). The two excel files in this folder represent the raw data and the detailed calculations of the vessel permeability in nude mice injected with undifferentiated and differentiated myofibroblast-like PDGFRα+ cells before and after hind limb ischemia. Contralateral skeletal muscles without hind limb ischemia and injection were used as controls. Folder Flow cytometry of PDGRα cells stromal markers with and without ischemia: This analysis was performed to characterize the expression of stromal markers in skeletal muscle PDGFRα+ cells. Flow cytometric analysis was performed in murine skeletal muscle obtained from WT mice at 10-12 weeks of age. PDGFRα+ cells were gated first as DAPI(-), then as PDGFRα+ cells (APC antibody) and finally with antibodies PE-conjugated antibodies for stromal markers (CD73, CD105, CD29, and PDGFRb). The experiment was done in not injured and injured (hind limb ischemia) mice. Folder Flow cytometry of isotype control for stromal markers with and without ischemia: This folder contains samples labeled with isotype control for the previous experiment. This analysis was also perfomed in uninjured and injured tissues.

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