Rosettesep reagent human cd45 depletion cocktail

Blood samples were voluntarily donated by healthy employees of Tosoh Corporation on the day of the experiments, and collected in Venoject II vacuum collection tubes supplemented with EDTA-2K (TERUMO, Tokyo, Japan).
In the spike-in experiments, 3.0 mL samples of blood were spiked with tumor cells, whose number was adjusted in advance by means of fluorescently labeling with Calcein Blue AM reagent to distinguish live from dead cells. Negative enrichment of the tumor cells was performed using the RosetteSep reagent Human CD45 Depletion Cocktail (STEMCELL technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol with some modification. Further information of procedure of negative enrichment of tumor cells can be found in the Supporting Information. As a result of the procedure, a suspension of tumor cell-enriched mononucleated cells with conductivity of < 200 μS/cm was obtained. The resulting number of mononucleated cells ranged from 1 ×10⁵ to 5 × 10⁵, depending on the donor’s blood condition.

All of the blood samples were collected before surgery, or before newly scheduled chemotherapy. Three mL of peripheral venous blood was collected and transferred to a 5 mL blood collection tube containing EDTA (TERUMO, Tokyo, Japan). In a Tosoh enrichment tube (Figure 1a), 3.5 mL Ficoll (GE Healthcare, Chicago, IL, USA), 3 mL blood sample, 3 mL fixative solution, and 180 μL Tirofiban were added and mixed well. After standing for 15 min at room temperature (RT), 75 μL of RosetteSep reagent Human CD45 Depletion Cocktail (STEMCELL technologies, Vancouver, BC, Canada) was added to deplete the white blood cells and the red blood cells. After centrifugation (2000× g, 10 min, 24 °C), the concentrated mononuclear layer was collected, mixed with a lysing solution, then centrifuged at 300× g for 10 min at 24 °C. After removing the supernatant, the pellet was resuspended in 30 mL of polyethylene glycol bovine serum albumin (PEG-BSA) and centrifuged at 300× g for 5 min at 24 °C. This procedure was repeated three times. After removing the supernatant, the final pellet was suspended in PEG-BSA.