Pan monocyte isolation kit

Monocytes and CD8⁺ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), which were obtained from the Korean Redcross Blood Services with the approval of the Institutional Review Board of Ajou University Hospital (AJIRB‐BMR‐SMP‐17‐424). The PBMCs were isolated using Ficoll Paque Plus (GE Healthcare Life Sciences, Buckinghamshire, UK). The monocytes were isolated using a pan monocyte isolation kit (MACS 130‐096‐537, Miltenyi Biotech, Gladbach, Germany), whereas the CD8⁺ T cells were isolated using a CD8⁺ T cell isolation kit (MACS, 130‐096‐495, Miltenyi Biotech). The monocytes and CD8⁺ T cells were maintained in complete Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum (FBS, GIBCO‐BRL, Grand Island, NY). To obtain activated T cells, the isolated primary CD8⁺ T cells with Dynabeads Human T‐Activator CD3/CD28 (Thermo Fisher Scientific, Inc., Waltham, MA) was incubated for 72 h and were used for migration assay.

This study involving human blood samples from healthy volunteers has been approved by the local ethics committee of the Goethe-University Frankfurt am Main. Oral consent was obtained and documented. Blood samples were obtained from four healthy donors and PBMC isolated via density gradient centrifugation with Ficoll Paque Plus (GE Healthcare, Chicago, IL, USA). Monocytes were enriched by negative selection using a pan monocyte isolation kit (MiltenyiBiotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. This isolation kit allows the enrichment of all three major blood monocyte populations, i.e. "classical" (CD14^highCD16^–), "non-classical" (CD14^lowCD16⁺) and "intermediate" (CD14^highCD16⁺) monocytes.

Frozen human peripheral blood mononuclear cells (PBMCs) were thawed, washed and pooled in MACS buffer (0.5% BSA in PBS containing 2 mM EDTA). Monocytes were isolated using negative selection, pan-monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s specifications. Briefly, PBMCs were resuspended in MACS buffer. FcR blocking reagent and biotin-antibody cocktail were added, mixed thoroughly and incubated at 4 °C. After 5 min incubation, MACS buffer and anti-biotin micro beads were added and incubated at 4 °C for 10 min. Stained cells were then washed with MACS buffer and pelleted (4 °C, 300 × g, 8 min). The pellet was then resuspended in MACS buffer and loaded onto the MACS column. The column was then washed twice with MACS buffer. The flow-through and washed fraction containing unlabelled monocytes was collected. Cell number and viability were determined by 0.2% trypan blue staining. Monocytes were cultured and stimulated in the presence of 1,2-¹³C₂-glucose using the protocol described above. Cells were then harvested, pelleted and shock-frozen in liquid nitrogen. A 100 µl of a mixture of acetonitrile and water (H₂O:ACN (1:1)) was then added to the frozen pellet and incubated on ice for 5 min. Cell lysate was then centrifuged at 15,000 × g for 10 min (4 °C). A 75 µl of supernatant was stored at −80 °C until HPLC-MS/MS analysis.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood by Ficoll-Paque (GE Healthcare, Illinois, USA) gradient centrifugation and monocytes (MNs) isolated using the Pan Monocyte Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), with >95% purity assessed by flow cytometry. MNs were plated onto Poly-D-lysine coated tissue culture plates (1.3 × 10⁵ cells/well) and rested overnight at 37°C/5%CO₂ in RPMI-1640 medium supplemented with 10% heat-inactivated human AB serum (Sigma Aldrich, Missouri, USA), 2 mM L-glutamine and 1 mM sodium pyruvate before infection. THP-1 cells (ATCC #TIB-202) were differentiated with 25 ng/ml PMA for 48 h and rested for 24 h prior to infection.

Monocytes from HCs and patients with cirrhosis expressing high (>20%/450 MFI) versus low (<7.5%/300 MFI) AXL levels were isolated using the Pan Monocyte Isolation Kit (Miltenyi Biotec) and co-cultured with allogeneic CD3⁺ T cells from a different healthy donor, isolated using the Pan T Cell Isolation Kit (Miltenyi Biotec), in a 1:1 ratio. T cell stimulation was induced with anti-CD2/CD3/CD28 beads (T cell Activation/Expansion Kit; Miltenyi Biotec) as previously described (20 (link)). T cells were stained with carboxyfluorescein succinimidyl ester at day 0. Proliferation was assessed at day 2 of co-culture by flow cytometry.

Frozen samples of PBMCs from Kenyan adults and children with acute P. falciparum malaria, as well as matched convalescent (P. falciparum PCR negative) samples acquired 28 days after treatment, were provided by Case Western Reserve. Monocytes were isolated from PBMCs using Pan Monocyte Isolation kit (Miltenyi Biotec, 130-096-537) following the manufacturer’s instructions.

Freshly obtained blood samples from healthy donors were provided by the Institute of Transfusion Medicine, Hannover Medical School. Informed donor consent was obtained, and the experiments were approved by the Hannover Medical School ethics committee in accordance with the Declaration of Helsinki. Monocytes were isolated as previously described [33 (link)] using the Monocyte Isolation Kit II or the Pan Monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated overnight before further use. The purity of isolated primary human monocytes was assessed by dual cell labeling for 30 min (4 °C, dark) using Alexa Fluor 405-CD45 (Invitrogen) and allophycocyanin-CD14 (BD Biosciences) recombinant human antibodies. Contamination with other leukocytes was excluded using the antibodies allophycocyanin-CD3, PE-CD19, FITC-CD56 (BD Biosciences), and FITC-CD15 (Invitrogen) [34 (link)].