Easysep direct human monocyte isolation kit

Human blood was collected as described above. Monocytes were isolated using an EasySep™ Direct Human Monocyte Isolation Kit (STEMCELL Technologies), which uses a series of negative-selection immunomagnetic labeling and subsequent magnetic separations to isolate monocytes directly from human whole blood. Then, monocytes were resuspended in IMDM with 20% FBS and stained with 0.25 μM CellTracker deep red (CTDR, ThermoFisher) for 45 min and 20 μg/mL Hoechst 33342 for 10 min. Monocytes were adjusted to a concentration of 0.6 million cells/mL for swarming experiments of monocytes alone and monocytes with neutrophils.

CD14^+/CD16^- cells were isolated by negative selection (EasySep™ Direct Human Monocyte Isolation Kit, STEMCELL Technologies, Vancouver, BC, Canada) from 40 ml of whole blood from volunteers (PLWH and HIV^- controls) and plated for M1 polarization and LPS stimulation. Cells were cultured for 6 days in RPMI/10% FBS/GM-CSF [25 ng/ml] then given LPS [100 ng/ml, Sigma Aldrich, St. Louis, MO] and IFNγ [20 ng/ml] in 5% FBS for 24 h, as we and others have reported (80 (link), 81 (link)). Hyclone Defined FBS was from Gibco (New York, NY). Human GM-CSF and IFN-γ were from R&D Systems (Minneapolis, MN). To mimic endotoxin tolerance, cells were exposed to a higher concentration of LPS [1 µg/ml; LPS challenge] for an additional 5 hours. Cell purity and macrophage polarization (development to M1) were assessed with flow cytometry, as we previously reported (81 (link)).

Human blood samples for isolation of monocytes, were collected with informed consent from healthy donors into EDTA by a certified phlebotomist according to the guidelines and approval of California University Long Beach Institutional Review Board. Human monocytes from 3 individual donors were isolated using the EasySep Direct Human Monocyte Isolation Kit according to the manufacturer’s instructions (StemCell). Cell purity was determined using the Scepter cell analyzer (EMD Millipore, Darmstadt, Germany) and monocyte populations used were greater than 90% pure. Isolated monocytes were cultured for 8 days in RPMI1640, 10% FCS, 2mM L-Glutamine and 1% penicillin/streptomycin containing 25 ng/ml rhM-CSF (Peprotech, Rocky Hill, NJ) to stimulate differentiation into human monocyte-derived macrophages (HMDM).

CD14 positive monocytes were purified from buffy coats purchased from The Blood Center (New Orleans, LA), using EasySep Direct Human Monocyte Isolation Kit for immunomagnetic negative selection (STEMCELL Technologies). Cells were seeded at 2x10⁶ cells/ml in serum-free CTL test media, incubated for 3 days with or without 10ug/ml LTA1 and had supernatants were collected.

Assays were performed on purified human classical monocytes isolated using immunomagnetic negative sorting (EasySep Direct Human Monocyte Isolation Kit, StemCell Technologies, Cambridge, MA). As we have previously described (46 (link)), this procedure results in a highly pure (> 85%) population of classical monocytes, with depletion of intermediate and non-classical monocytes due to the presence of an anti-CD16 antibody in the cocktail. Isolation purity was verified at several points throughout the current study and averaged approximately 90% (not shown). Cells were counted at 10× dilution using a Scepter cell counter (Millipore Sigma, St. Louis, MO). Isolated monocytes were immediately utilized in downstream assays, and no cells were frozen for later use.

Lenti-X 293T cells (Takara) were cultured at 0.5–1 × 10⁶ cells/ml in Dulbecco’s modified Eagle’s Medium (DMEM) GlutaMax-I supplemented with 1X penicillin/streptomycin (PS) and 10% fetal bovine serum (FBS) (Gibco). NLRP3-deficient U937 cells have been previously described (Lagrange et al., 2018 (link)). U937 and reconstituted U937 NLRP3-deficient cells were cultured at 0.25–1 × 10⁶ cells/ml in Roswell Park Memorial Institute (RPMI) 1640 GlutaMax-I supplemented with 1X PS and 10% FBS (Gibco). Monocytes were purified from 10 to 20 ml of blood samples (drawn the day before in heparin tubes) using EasySep Direct Human Monocyte Isolation kit (StemCell) and cultured at 0.07–0.15 × 10⁶ cells/ml in RPMI-1640 GlutaMax-I supplemented with 1X PS and 10% FBS (Gibco). The purity of the monocyte fractions was assessed by anti-CD45-APCR700 and anti-CD14-PE-CF594 (BD Horizon) staining and FACS analysis, and ranged between 66 and 82% CD45+/CD14+ (gated on total cells) and 81–95% (gated on CD45%).

Monocytes have been extensively employed in the study of trained immunity (14 (link), 24 (link)). Whole blood containing EDTA was purchased from BioIVT (Gray, Tennessee). The blood samples were de–identified except for age, gender, and race. BioIVT provided blood that was collected from healthy individuals in 3 age groups, i.e. 20–30, 31–59, >60 years of age. There were 35 individuals in each age group and a total of 105 individuals in the study. The age range for the entire cohort was 20–71 years. A detailed description of the subjects is provided in Table 1. Monocytes were isolated from whole blood using immunomagnetic negative selection with the EasySep™ Direct Human Monocyte Isolation Kit (StemCell #19669). Monocyte purity was assessed by flow cytometry. This method yields CD14+ monocyte purity of up to 97.1%.