Pe cy7 conjugated anti cxcr4

Peripheral blood PC were analyzed for the expression of surface antigens using direct flow cytometry (BD FACS Canto II Flow Cytometer). Three hundred µl of venous blood (anticoagulant: EDTA) was incubated with fluorochrome-labeled monoclonal mouse anti-human antibodies, namely, FITC-CD34 (BD Biosciences), PE-VEGF2R (R&D system - also known as “Kinase insert Domain Receptor-KDR”), APC-CD133 (Miltenyi) and PE-Cy7-conjugated anti-CXCR4 (EBioscience, clone 12G5 for 15 minutes. Red blood cells were removed by lysis in 1.5 ml of ammonium chloride lysis buffer which was added to the sample and incubated for an additional 10 minutes. The lysis process was stopped by adding 1.5 ml of staining medium (PBS with 3% heat-inactivated serum and 0.1% sodium azide) was added to stop lysing. Up to five million events were acquired from the Cytometer with Flowjo software (Treestar, Inc.) used for subsequent analysis of accumulated data. List mode files containing at least 3,000,000 events were collected so that analysis of rare sub-populations would contain an adequate number of events. Absolute numbers of each cell subset per milliliter were determined by multiplying the counts with the number of monocytes per milliliter of blood. (Online figure I)

Peripheral blood mononuclear cells were analyzed for the expression of surface antigens using direct flow cytometry (BD FACS Canto II Flow Cytometer; BD Biosciences, San Jose, CA) as described previously. ^{14 (link),15 (link)} Three hundred μl of venous blood (anticoagulant: EDTA) was incubated with fluorochrome-labeled monoclonal mouse anti-human antibodies, namely, FITC-CD34 (BD Biosciences), PE-VEGF2R (R&D system - also known as “Kinase insert Domain Receptor-KDR”), APC-CD133 (Miltenyi) and PE-Cy7-conjugated anti-CXCR4 (EBioscience, clone 12G5 for 15 minutes. Red blood cells were removed by lysis in 1.5 ml of ammonium chloride lysing buffer which was added to the sample and incubated for an additional 10 minutes. The lysis process was stopped by adding 1.5ml of staining medium (PBS with 3% heat-inactivated serum and 0.1% sodium azide). Five million events were acquired from the Cytometer with Flowjo software (Treestar, Inc.) used for subsequent analysis of accumulated data. List mode files containing at least 3,000,000 events were collected so that analysis of rare sub-populations would contain an adequate number of events. Absolute numbers of each cell subset per milliliter were determined by multiplying the counts with the number of monocytes per milliliter of blood.