Straightfrom whole blood cd14 microbeads

Primary human macrophages were generated from peripheral blood mononuclear cells as previously described (16 (link)). Briefly, leukocyte reduction chambers were obtained from healthy human donors from discarded apheresis products via the Crimson Core Biobank. Monocytes were labeled using StraightFrom Whole Blood CD14 MicroBeads (Miltenyi Biotec) and purified using an autoMACS Pro Separator (Miltenyi Biotec). Monocytes were then cultured in IMDM (Thermo Fisher Scientific) supplemented with 10% ultra-low IgG fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 292 μg/mL L-glutamine, and 20 ng/mL human M-CSF (Peprotech) for at least 7 days. Cells were passaged or replated as necessary and typically maintained in culture for 2–4 weeks.

CD14⁺ monocytes were isolated using the positive immunomagnetic selection kit from Miltenyi Biotec according to the manufacturer’s instructions. A total of 2 mL of whole blood and 100 µL of StraightFrom Whole Blood CD14⁺ MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were incubated at 4 °C for 15 min. Then 3 mL of phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA), and 0.4% EDTA was added and the mixture was spun at 450 g for 10 min. Plasma was aspirated, and another 1 mL of buffer was added. The cell suspension was loaded onto an LS magnetic column (Miltenyi Biotec, Bergisch Gladbach, Germany) placed in the magnetic field of a MACS Separator (MIDIMACS) and rinsed three times with buffer. The column was then removed from the magnetic field, and the CD14⁺ fraction was eluted in a total volume of 5 mL. Cell concentrations were taken to ensure accurate and sufficient cell count using a Countess II FL Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Cell counts in the range of 1.0 × 10⁵ were considered accurate and acceptable. The fraction was then centrifuged at 450× g for 10 min, elution buffer was aspirated, and the cells were lysed with 600 µL of Buffer RLT (Qiagen, Hilden, Germany; catalog #79216). Lysate was then frozen at −80 °C until RNA was isolated.

Whole blood samples from all participants were collected in EDTA-containing tubes. All plasma samples were separated by centrifugation within 4 h of collection to ensure constant preanalytical conditions. PBMC were isolated by Ficoll–Paque density gradient centrifugation. CD14+ cells were labelled with magnetic StraightFrom Whole Blood CD14 MicroBeads and then separated using a Whole Blood Column Kit (both Miltenyi Biotec Inc., Gladbach, Germany). Plasma samples and cell culture samples were stored at − 80 °C. PBMC and freshly isolated monocytes were snap-frozen and stored at − 80 °C. No freeze–thaw cycles occurred before use.