K3edta vacutainer tubes

The peripheral blood samples were collected from the cubital vein using K3EDTA Vacutainer tubes (Greiner Bio-One, Kremsmünster, Austria). Plasma was prepared by two-step centrifugation of 6 mL of blood at 950 rcf at 10 min at 4 °C and then at 11,000 rcf at 10 min at 4 °C–to remove any cell debris as a standard procedures used for cell-free DNA assessment.
For CEA detection, the blood serum was separated by centrifugation at 1700 rcf for 10 min from 4 mL of blood collected in Vacuette^® blood collection tubes (Greiner Bio-One, Kremsmünster, Austria). Until analysis plasma and serum samples were stored frozen at −80 °C.

Basophils for the rolling assay were isolated according to the manufacturer’s instructions by negative selection using the Human Basophil Enrichment Kit (Stemcell). Briefly, approximately 40 ml of whole blood was collected in K₃EDTA vacutainer tubes (Greiner). Erythrocytes were first depleted using HetaSep (Stemcell) to obtain the nucleated cells, which were then incubated with the Human Basophil Enrichment Cocktail for 10 min at room temperature. The suspension was mixed with EasySep Nanoparticles at room temperature for 10 min. The cell suspension was added to a total volume of 10 ml and mixed by pipetting up and down 3–4 times. The tube containing the cell suspension was placed in the Silver EasySep Magnet and basophils were isolated by pouring. The number of basophils typically range from 3.6 × 10⁵ to 4.5 × 10⁵ and the purity of the basophil was determined to be 96.5–98.2%.

Whole blood was collected in 9 ml K₃EDTA Vacutainer Tubes (Greiner Bio-One, Kremsmünster, Austria). Peripheral blood mononuclear cells (PBMC) were isolated from fresh venous blood taken from 5 healthy adult volunteers at the National University of Singapore and at the Singapore Immunology Network, Agency for Science Technology and Research (Table 1B). PBMC were isolated from the buffy coat by density gradient centrifugation with Lymphoprep (Stemcell, Axis-Shield PoC AS, Oslo, Norway), followed by PBS washes until the supernatant was clear. We obtained a 98% viability of PBMC, as assessed by Trypan blue staining. PBMC were suspended at a density of 4 × 10⁶/ml in complete media (RPMI supplemented with 10% heat-inactivated FBS, 1% L-glutamine, 1% penicillin-streptomycin). One of the PBMC donors had to be excluded after quality control post-hoc analyses. NK cells were isolated by negative selection from PBMC using EasySEp Human NK Cell Enrichment Kit (Stemcell Technologies). The purity of the isolated NK cells was 84.4% of CD45+ cells.