The enriched thymic stromal fraction was centrifuged at 1,500 rpm and resuspended in 1X PBS with 0.5% bovine serum albumin and 2mM EDTA prior to antibody staining. Cells were incubated with fluorochrome-conjugated antibodies against CD3, CD4, CD8, TER-119 (Biolegend) and CD45 (BD Biosciences) to exclude remaining thymocytes and red blood cells (“Hem+” cells), and anti-CD326 (EpCAM), anti-Ly-51, anti-CD140a (PDGFRα), anti-CD146 (Mel-CAM) and anti-CD31 (PECAM-1) (Biolegend) for stromal cell analysis. Cells were analyzed on the LSRII Fortessa (BD Biosciences) and isolated using the FACS AriaII (BD Biosciences). All gating was performed following exclusion of doublets (SSC-H/SSC-W and FSC-H/FSC-W) and dead cells using 4’,6-diamidino-2-phenylindole dihydrochloride DAPI (Thermo Fischer). Data was analyzed using FlowJo software (Tree Star).
Anti cd31 pecam 1
Manufactured by BioLegend
Anti-CD31 (PECAM-1) is a monoclonal antibody that binds to the CD31 (PECAM-1) protein, which is expressed on the surface of endothelial cells, platelets, and some types of leukocytes. CD31 is involved in cell-cell adhesion and plays a role in various cellular processes.
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Lab products found in correlation
2 protocols using anti cd31 pecam 1
1
Thymic stromal cell isolation and analysis
2
Multicolor Flow Cytometry of Cell Surface Markers
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The cells were prepared, fixed and incubated with FITC labelled anti-CD31 PECAM-1 (Biolegend Part Number 303104), Alexa Fluor® 488 anti-CD54 ICAM-1 (Biolegend Part Number 322714) and Alexa Fluor® 647 anti-CD62E E-Selectin (Biolegend Part Number 148801) according to the manufacturer’s instruction. Flow cytometry was performed using a BD FACS Calibur. All analyses were performed using FlowJo software (Tree Star Inc., 92 Ashland, OR, USA). Cytometry data was gated to remove cell debris and doublets. Cells defined to be expressing the antibody were defined as FITC positive and the percentage of FITC positive cells in each of the test subpopulations was determined.
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