RosetteSep CD45 Depletion, 10mL

This immunodensity depletion cocktail from STEMCELL Technologies is designed to enrich epithelial circulating tumor cells (CTCs) from human whole blood by depleting CD45+ cells. The product is a combination of monoclonal antibodies in PBS, including a mouse monoclonal IgG1 directed against human and chimpanzee CD45 (and, according to a third-party source, also targeting CD66b and glycophorin A on red blood cells). The cocktail crosslinks unwanted hematopoietic cells to multiple red blood cells, forming immunorosettes that pellet along with free red blood cells during centrifugation over a density gradient medium. The desired, antibody-unlabeled cells are collected as a highly enriched population at the interface between the plasma and the density gradient medium and are immediately available for downstream applications such as flow cytometry, cell culture, or DNA/RNA extraction. The protocol requires no special equipment or training and can be combined with SepMate tubes for consistent, high-throughput sample processing.

For optimal performance, whole peripheral blood should be used within 24 hours of collection and stored at 15 - 25°C, with nucleated cell concentrations not exceeding 5 x 10^7 cells/mL. The cocktail is added at 50 μL/mL of sample, mixed, and incubated at room temperature for 20 minutes (standard tube) or 10 minutes (SepMate). The sample is then diluted with an equal volume of Dulbecco's PBS containing 2% fetal bovine serum and layered over a density gradient medium such as Lymphoprep (density 1.077 g/mL). Centrifugation is performed at 1200 x g for 20 minutes with brake off using a standard tube, or 1200 x g for 10 minutes with brake on when using SepMate. The cocktail is stored at 2 - 8°C, should not be frozen, and remains stable until the expiry date on the label; components may be shipped at 15 - 25°C, and a precipitate may be observed in the vial but will not affect performance. According to the manufacturer's example data, when CAMA epithelial tumor cells were seeded into whole blood at a starting frequency of 1.8% (Ber-EP4+CD45-), the resulting CD45-depleted fraction contained 83% CAMA cells with a 3.6 log depletion of CD45+ cells. This configuration of the product is intended for processing 200 mL of whole blood.

STEMCELL Technologies' products are for research use only unless otherwise stated, and the company notes that its quality management system is certified to ISO 13485.