UCBMC were obtained by standard density gradient centrifugation over Ficoll (BD Bioscience, San Jose CA), resuspended in 10% DMSO/FBS, frozen using Mr. Frosty Freezing Containers (Thermo Fisher, Waltham MA), and stored in liquid nitrogen until analysis. Frozen UCBMCs were thawed and UCB monocytes were purified using CD14 antibodies conjugated to magnetic microbeads per manufacturer’s recommendations (Miltenyi Biotech, San Diego CA). Magnetically bound UCB monocytes were washed and eluted for collection. Positive selection of UCB monocytes was chosen to ensure high purity of the population, which was assessed using flow cytometry and was ≥90% for all samples analyzed.
Cd14 antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
CD14 antibodies are laboratory reagents used to detect and analyze the CD14 protein, which is expressed on the surface of certain immune cells, such as monocytes and macrophages. These antibodies can be used in various immunological and cell biology applications, including flow cytometry, immunohistochemistry, and cell isolation.
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Lab products found in correlation
Magnetic microbead, by Miltenyi Biotec (3 mentions)
Ficoll, by BD (1 mentions)
Mr frosty freezing container, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ apc, by BioLegend (1 mentions)
Anti cd56 fitc, by BioLegend (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Anti cd16 pe, by BioLegend (1 mentions)
Cfp 10, by BEI Resources (1 mentions)
Esat 6, by BEI Resources (1 mentions)
Anti cd27 pe, by BD (1 mentions)
Automacs device, by Miltenyi Biotec (1 mentions)
Tc 20 cell counter system, by Bio-Rad (1 mentions)
Interleukin il 4, by R&D Systems (1 mentions)
Granulocyte macrophage colony stimulating factor, by R&D Systems (1 mentions)
8 protocols using cd14 antibody
1
Isolation of UCB Monocytes
2
Splenic CD14+ Macrophage Isolation
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Splenic CD14+ macrophages were purified from freshly thawed splenic leukocytes using CD14 antibodies conjugated to magnetic microbeads per the manufacturer's recommendations (Miltenyi Biotec, San Diego CA). Positive selection of macrophages was chosen to ensure high purity of the population, which was assessed using flow cytometry and was on average ≥ 90%.
3
Macrophage Polarization and RCT001 Treatment
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Human peripheral blood samples were obtained from healthy donors and the monocytes were enriched with CD14 antibodies (Miltenyi, 130–050-201) by positive selection. The purified human monocytes were cultured in RPMI 1640 medium with Glutamax-I supplemented with 10% FBS. To induce macrophage differentiation, the purified monocytes were stimulated for 5 days with colony-stimulating factor 1 (CSF-1) to generate M0-like macrophages. To induce polarization into different macrophage phenotypes, the M0-like macrophages were subjected to specific treatments. For M1-like polarization, lipopolysaccharide (100 ng/ml) and interferon gamma (20 ng/ml) were administered to M0-like macrophages for 48 h. M2-like polarization was achieved by delivering IL-4 (20 ng/ml) to M0-like macrophages for 48 h. After polarization, M0-, M1- and M2-like macrophages were treated with RCT001 at a concentration of 2.5 µM applied 48 h after polarization. Further information can be found in the Supplementary methods .
4
Detailed Multiparametric Immunophenotyping Protocol
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For flow cytometry, we used anti-CD3 FITC (BioLegend, USA, Cat#300440) and PerCP (BD Biosciences, USA, Cat#347344), anti-CD56 FITC (BioLegend, USA, Cat#318304) and APC (BD Biosciences, USA, Cat#555518), anti-CD16 PE (BioLegend, USA, Cat#302008); for staining memory cells anti-KLRG1 FITC (BioLegend, USA, Cat#138410), anti-CD27 PE (BD Biosciences, USA, Cat#555441); for intracellular staining anti-IL-32α (R&D Systems, USA, Cat#IC30402A) and anti-IFN-γ APC (BioLegend, USA, Cat#502512) antibodies were used. Magnetic beads conjugated to anti -CD56 (Miltenyi Biotec, Germany, Cat# 120-000-307) and -CD14 antibodies (Miltenyi Biotec, Germany, Cat# 120-000-305) were used for positive selection of NK cells and monocytes respectively.
For in vitro stimulation assays, we used ESAT-6 (BEI Resources, USA, Cat#NR-50711) and CFP-10 (BEI Resources, USA, Cat#NR-50712) peptide pools consisting of 21 and 22 individual peptides belonging to 6-kDa ESAT-6 and 10-kDa CFP-10 respectively. γ-irradiated Mtb H37Rv whole cells were used for some experiments (BEI resources, USA, Cat#49098).
For in vitro stimulation assays, we used ESAT-6 (BEI Resources, USA, Cat#NR-50711) and CFP-10 (BEI Resources, USA, Cat#NR-50712) peptide pools consisting of 21 and 22 individual peptides belonging to 6-kDa ESAT-6 and 10-kDa CFP-10 respectively. γ-irradiated Mtb H37Rv whole cells were used for some experiments (BEI resources, USA, Cat#49098).
5
Magnetic Separation of Monocytes
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Monocytes were purified from freshly thawed PBMC using CD14 antibodies conjugated to magnetic microbeads per the manufacturer’s recommendations (Miltenyi Biotec, San Diego, CA). Magnetically bound monocytes were washed and eluted for collection. Purity was assessed using flow cytometry and was on average ≥95% (Figure S7 A).
6
Isolation and Characterization of Human CD14+ Cells
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Fresh human peripheral blood (n = 6) was collected with an approved IRB and written consent from donors from The Ohio State University Medical Center, Columbus, OH and processed following earlier described protocol.11 , 19 In, brief, peripheral blood mononuclear cells were isolated from freshly collected blood using Ficoll‐Paque density centrifugation. CD14+ cells were isolated by using an AutoMACS device, CD14+ antibody and reagents (all from Miltenyi Biotec, San Diego, CA, USA) following earlier established protocol.11 , 20 A part of the CD14+ cells was used for RNA extraction, and another part was subjected to protein extraction.
7
Isolation and Purification of Monocytes from PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors at the blood bank of the National Institute of Respiratory Diseases in Mexico City. PBMCs were collected and counted to determine their viability using the TC20 cell counter system (Bio-Rad, California, USA). Monocytes were enriched by employing positive selection with magnetic microbeads coated with CD14 antibody (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity was confirmed by flow cytometry, and the purified cells were found to be 90%–95% of the intended cell type.
8
Monocyte-Derived Dendritic Cell Generation
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Following in vitro experiments, monocyte-derived dendritic cells (Mo-DCs) were used as an alternative to primary peritoneal DCs ( 16), owing to the technical limitations in isolating and culturing pure primary peritoneal DCs. Mo-DCs were prepared as reported previously (17, 18) . Briefly, peripheral blood was obtained from healthy donors and peripheral blood mononuclear cells were separated by means of density gradient centrifugation with Ficoll-Paque. Monocytes were purified with the use of magnetic microbeads with CD14 antibody (Miltenyi Biotec) and plated at 5 Â 10 6 /mL in RPMI 1640 medium with 10% FBS, 20 ng/mL granulocyte-macrophage colony-stimulating factor (R&D Systems) and 20 ng/mL interleukin (IL) 4 (R&D Systems). Mo-DCs were obtained after 4 days of culture.
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