Cells were labelled with BD™ DilC12(3) (BD Biosciences), suspended in X-Vivo 10 (Lonza, http://www.lonza.com ), then seeded (106 cells/w) in BD Biocoat™ Matrigel™ invasion chambers as well as control inserts (BD Biosciences) in 24 well plates; complete CFU-Hill liquid medium (StemCell Technologies) was added to the wells, below the inserts. After 24 hours at 37°C, 5% CO2, the fluorescence of invading cells was determined by a bottom fluorescence plate reader (Infinite F200, Tecan, http://www.tecan.com ). The Invasion Index was calculated as follows:
Cfu hill liquid medium
Manufactured by STEMCELL
Sourced in Canada
CFU-Hill liquid medium is a pre-made culture medium designed for clonogenic assays. It supports the growth and enumeration of hematopoietic progenitor cells in the colony-forming unit (CFU) assay.
Automatically generated - may contain errors
3 protocols using cfu hill liquid medium
1
Cell Invasion Assay Using Matrigel
2
Isolation and Enumeration of CFU-Hill Colonies
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CFU-Hill colonies were cultured from PBMCs to isolate putative early EPCs. PBMCs were isolated from anticoagulated whole blood by density gradient centrifugation in Histopaque-1077 (Sigma Aldrich). Cells were first seeded on 12-well fibronectin-coated plates (Day 0) at 2.5×106 cells/well in CFU-Hill Liquid Medium (Stem Cell Tech). Non-adherent cells were then collected and replated onto 24-well fibronectin-coated plates at 1.0×106 cells/well (Day 2). CFU colonies were next scored as a central core of round cells surrounded by radiating spindle-shaped endothelial-like cells (Day 5) (Fig. 1A, B ). Following day 5 in culture, CFU-Hill colonies were counted, and mean colony number calculated.
3
Quantifying Endothelial Progenitor Cells
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The number of EPCs was measured using the method developed in the previous study with some modifications33 (link). Peripheral blood mononuclear cells were isolated using density-gradient centrifugation with Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) from 10 mL of whole blood and an ethylenediaminetetraacetic acid additive. One million mononuclear cells were suspended in a CFU-Hill liquid medium (#5900, Stemcell Technologies, Vancouver, British Columbia, Canada) in a fibronectin-coated 24-well plate (BD Biosciences). After 5–7 days of culture, the EC-CFUs were manually counted using light microscopy (Olympus BX-51, Olympus, Tokyo, Japan).
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