Direct human neutrophil isolation kit

To generate white blood cells (WBC), red blood cells were lysed from whole human blood, which was collected using either ethylenediaminetetraacetic acid (EDTA) or acid citrate dextrose (ACD) as a coagulant. Blood was mixed at a 1:10 ratio with ammonium-chloride‑potassium (ACK) lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.4) and incubated for 5 min at room temperature. The WBCs were pelleted by centrifugation (500 ×g, 5 min) at room temperature and then washed with cold phosphate-buffered saline (PBS). For the isolation of human neutrophils, whole blood was mixed with hetasep (Stemcell) in a 1:5 ratio and incubated for 30 min to separate leukocytes from erythrocytes. Subsequently, the straw-colored layer of leukocytes was transferred to a fresh tube and neutrophils were isolated using the direct human neutrophil isolation kit (Stemcell) according to manufacturer's instructions. Both WBCs and purified neutrophils were finally diluted in complete R10 media (RPMI-1640 media (Sigma) with 10% FBS, 2 mM L-Gluthamine and 100 U/ml penicillin/streptomycin) for each assay.

For studies of mechanisms of 2E9IgA1 modulation of infection, beads or M. tuberculosis bacilli were used with human macrophage/monocyte cell lines THP-1 and U937 (ATCC, LGC Standards, Teddington, UK), or neutrophils from blood of healthy volunteers, following ethical approvals. Neutrophils were purified from blood using HetaSep (Stemcell, Cambridge, UK) in a 1:5 ratio and incubated for 30 min to separate leukocytes from erythrocytes. Subsequently, the white-coloured layer of leukocytes was transferred to a fresh tube and neutrophils were isolated using the direct human neutrophil isolation kit (Stemcell) according to manufacturer’s instructions.

For the isolation of primary human neutrophils from healthy donor blood leucocytes were separated from erythrocytes by mixing the blood in a 5:1 ratio with Hetasep (Stemcell Technologies). After an incubation of 15 min at 37°C the leucocytes were washed (500xg, 10 min, 4°C) with MACS buffer (PBS, pH 7.2 + 0.5% BSA + 2mM EDTA, sterile filtered) and purified using the direct human neutrophil isolation kit (Stemcell Technologies). Purified neutrophils were suspended in R10 at density of 2.5x10⁵ cells/ml and used immediately for experiments. Human B cells from individuals of the EBV-infected cohort were isolated from frozen PBMCs after DNAseI treatment by negative selection using the EasySep human B cell isolation kit (Stemcell Technologies).