To analyse tumour blood vessel leakage, Hoechst 33258 dye (4 µg/ml; Sigma, H33258) was used. Hoechst dyes diffuse quickly from vessels and bind to the DNA of cells surrounding blood vessels, allowing for quantification of areas of uptake by perivascular tumour cells and hence blood vessel leakage (Janssen et al., 2005 (link)). Briefly, mice were injected sequentially with 100 μl phycoerythrin (PE)-conjugated CD31 (Biolegend; to stain functional blood vessels) followed 9 min later with 100 μl Hoechst 33258 (4 µg/ml; Sigma, H33258) via the tail vein and were killed 1 min later. Tumours were excised and snap-frozen. A total of 8–10 fields at ×20 magnification were analysed by ImageJ. For quantification, blood vessel leakage was calculated by counting the numbers of Hoechst 33258-positive nuclei surrounding PE–CD31-positive tumour areas. Results are shown in arbitrary units (AU).
Pe conjugated cd31
Manufactured by BioLegend
Sourced in United States
PE)-conjugated CD31 is a flow cytometry reagent that labels the CD31 (platelet endothelial cell adhesion molecule-1) protein. CD31 is expressed on the surface of endothelial cells, platelets, and some leukocytes. The PE (phycoerythrin) fluorophore is conjugated to the CD31 antibody, allowing the detection and quantification of CD31-positive cells by flow cytometry.
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2 protocols using pe conjugated cd31
1
Quantifying Tumour Blood Vessel Leakage
2
Isolation and Characterization of GFP-Labeled Mesenchymal Stem Cells from Synovium
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1 Â 10 6 GFP þ MSCs were injected 1week after the ACLT, then synovium was harvested 1day after the injection, following digestion with collagenase V for three hours. After filtering through a 70 mm cell strainer and centrifuging at 1500 rpm for 5 min, cells were suspended in ice-cold Hank's balances salt solution and then stained for 30 min on ice with a monoclonal antibody of APCconjugated CD90 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Synovial fluid was also extracted from the same knee, and prepared in the same manner without collagenase digestion. Propidium iodide (PI) fluorescence was measured, and a live cell gate was defined that excluded the cells positive for PI. Additional gates were defined as positive for GFP and CD90. Double positive cells were further analyzed for PE-conjugated CD29, PEconjugated CD31, and PEcy7-conjugated CD45 (Biolegend, San Diego, CA, USA). Flow-cytometric analysis and sorting were performed on a MoFlo (Beckman Coulter, Brea, CA, USA), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA) 26 .
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