Mojosort human cd14 selection kit

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation on Pancoll (PAN Biotech) according to standard laboratory protocols. Next, human primary monocytes were isolated from PBMCs by using magnetic nanobeads and the MojoSort Human CD14 Selection Kit (BioLegend) as described earlier (Tantawy et al., 2022 (link)). Subsequently, purified primary CD14-positive (CD14⁺) monocytes were resuspended in RPMI 1640 medium containing 10% hi-FBS and 1% penicillin‐streptomycin and used for cytotoxicity assays. Alternatively, purified CD14⁺ monocytes were differentiated into human monocyte-derived macrophages (HMDMs) in the same medium supplemented with 50 ng/ml of human macrophage colony stimulating factor (hM-CSF; Genscript). HMDMs were used at day 7 post-differentiation for cytotoxicity experiments.

Bone marrow derived macrophages (BMDM) were derived from isolated monocytes from the marrow of C57BL/6 mice using EasySep Mouse CD11b Positive Selection Kit II (Stemcell Technologies). Alternatively, human monocytes were isolated from discarded de-identified blood donation leukopaks from the Children’s Hospital Colorado Blood Donor Center, after written informed consent in accordance with the Declaration of Helsinki. Leukopak peripheral blood mononuclear cells (PBMCs), obtained using Lymphoprep (Stemcell Technologies), underwent CD14⁺ isolation using MojoSort™ Human CD14 selection kit (Biolegend). Monocytes were matured to macrophages on non-TC treated plates for three to seven days in cRPMI supplemented with 25ng/mL GM-CSF and 5ng/mL M-CSF, after which non-adherent cells were discarded. PtdSer expression on AML cell lines was induced via UV light exposure (15 minutes), followed by incubation at 37° for two to four hours, and then opsonized with 250nM recombinant mGAS6 (R&D Systems). Macrophages were pretreated with 300nM MRX2843 or vehicle for one hour in 24 well plates before AML cells were added (1.0×10⁶ cells/well). After 48 hours, non-adherent AML cells in the supernatant were removed; macrophages were washed with PBS thrice to remove residual AML cells.

PBMC-derived monocytes were isolated with the MojoSort Human CD14 Selection kit (BioLegend, San Diego, CA, USA), according to the manufacturer’s protocol. CD14+ cells (purity > 98% assessed by flow cytometry) were plated at 0.3 × 10⁶ cells/cm² in complete RPMI 1640 medium supplemented with 1000 U/mL rhGM-CSF (Peprotech, London, UK) and 250 U/mL rhIL-4 (Peprotech, London, UK) and cultured for six days at 37 °C with 5% CO₂, as previously described. Immature dendritic cells (DCs) were activated with 50 ng/mL lipopolysaccharide (LPS, from E. coli O127:B7, Sigma-Aldrich, St. Louis, MO, USA) in the same medium, at 37 °C, 5% CO₂, for two additional days. On days 6 and 8, DCs were analyzed for their expression of CD14, CD209, HLA-DR, CD80 and CD83 by flow cytometry.

CD14+ cells were isolated magnetically from PBMCs using the MojoSort™ Human CD14 Selection Kit (BioLegend), according to the manufacturer’s protocol. The cells were grown in 24-well plates (Corning) in RPMI-1640 medium (Sigma-Aldrich) supplemented with 1% Pen/Strep (Sigma-Aldrich), 10% heat-inactivated FBS (Sigma-Aldrich) (complete RPMI), 50 ng/ml GM-CSF, and 1,000 U/ml IL-4 (PeproTech). On days 2 and 4 of culture, the medium was replaced with fresh complete RPMI and cytokines, and the cells were harvested on day 7. To generate mature monocyte-derived dendritic cells (moDCs), LPS (Sigma-Aldrich) was added at 1 μg/ml on day 6. moDCs were incubated with 10 µg/ml of sEVs measured by protein concentration with NanoDrop 2000 (Thermo Fisher Scientific) overnight, and their marker expression was analyzed by flow cytometry.

Whole blood from apheresis was obtained from healthy donors at Boston Children’s Hospital’s (BCH) Plasma Donation Center. Because donor samples are completely de-identified at BCH prior to handling by research personnel and use in the laboratory, there is no IRB associated with the protocol. PBMCs were isolated via Ficoll gradient (Lymphoprep, StemCell 07801). CD14+ monocytes were then isolated from PBMCs using the MojoSort Human CD14 Selection Kit (Biolegend 480025) according to manufacturer protocol and plated at 5 × 106 cells per well in a 6-well plate in RPMI supplemented with 10% (v/v) FBS, penicillin/streptomycin, and 50 ng/ml M-CSF. On days 2, 4, and 6 after plating, culture media was refreshed. On day 6, cells were lifted from the plate using cold PBS and Accutase cell detachment solution (StemCell 07920) and replated at 2.5 × 104 cells/well in a 96 well plate.
On day 7, cells were given fresh media and treated with either nothing, 100 ng/ml LPS (Sigma–Aldrich, L2654), 20 ng/ml IL-4 (Peprotech, 200-04), or 100 μg/ml PAS polymers or β-glucans. After 24 h of incubation, cells were lifted from the plate with cold PBS and Accutase cell detachment solution and stained for CD68 (StemCell 60105FI) and either CD80 (Biolegend 333611), CD163 (Biolegend 305221), or CD206 (R&D Systems FAB25342P). Cells were then analyzed via flow cytometry (Attune, ThermoFisher Scientific).

PBMCs were separated from buffy coat samples with Lymphoprep (STEMCELL Technologies, Vancouver, BC, Canada). CD14⁺ cells were isolated by the MojoSort™ Human CD14 Selection Kit (BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol. Cells were seeded in 24-well plates in complete RPMI medium (supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin + 100 µg/ml streptomycin) at a density of 1 x 10⁶ cells in 1 ml of medium per well. Cells were cultured for 7 days at 37°C and 5% CO₂ in the presence of 50 ng/mL (500 U/mL) GM-CSF and 200 ng/mL (1000 U/mL) IL-4 for the generation of immature monocyte-derived dendritic cells (iMDDCs). Cytokine-supplemented medium was replaced on day 2 and 4 of the culture. To generate mature monocyte-derived dendritic cells (mMDDCs) 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) was added on day 6 of the culture.