Hbss without calcium or magnesium

Within 20 minutes post-collection, the CD15+ cells (comprising neutrophils, scarce eosinophils, and a rare subtype of T cells) were isolated from the EDTA-treated blood sample using anti-human CD15 antibody-coated Dynabeads (Invitrogen) with minor modifications to the manufacturer’s protocol. Immediately before use, the beads were re-suspended and washed as recommended. The bead isolation buffer (BIB) was modified by using HBSS without calcium or magnesium (Gibco). Blood volumes of 750 μl were used in 1.5 mL tubes. When larger volumes were processed, multiples of 750 μl were used. Instead of the recommended initial wash steps, the blood was spun at 3000 rpm (604 g) for 10 minutes, the plasma removed, and 750 μl of BIB was added just before adding the beads. Each wash step used a 1:1 volume to the original blood (typically 750 μl BIB), to keep a 1x concentration of the CD15+ cells. Cell capture and washing were performed using the MAGNA-SEP magnet (Invitrogen) for two minutes. Cell counts of the 1x isolate were performed using 10 μl in a hemocytometer at 200X using phase contrast. The identities of the isolated cells were confirmed by fixing with 4% formaldehyde in 1x PBS, and using a May-Grünwald stain (Wright-Giemsa stain with eosin Y).

To analyze the tumor immune microenvironment during the anticipated efficacious period of immune checkpoint activity, tumors were collected at day 14–17 after starting immune checkpoint inhibitor. Fresh tumors were dissociated into single cell suspensions with DNAse I (Invitrogen), collagenase Type IV (Sigma), and hyaluronidase (MP Biomedicals) for 1 h at room temperature using a dissociator (Miltenyi) with gentleMACS C-tubes. To remove calcium, cells were resuspended for 5 min in HBSS without calcium or magnesium (Gibco), then resuspended in 5 mM of EDTA for 30 min at room temperature. Next, cells were passed through a 70 μm filter before ACK lysing buffer (KD Medical Inc) was added to remove red blood cells before flow cytometry. Immediate staining was performed for surface marker expression to analyze with flow cytometry.