To polarize human macrophages, monocyte derived macrophages were plated in 48 well plates at 5 × 104 cells per well in 500μl RPMI medium and allowed to rest 2hours at 37°C before treat of cytokines or LPS. The human M0 macrophages were either left untreated or treated for 48h to induce macrophage polarization: (i) for M1 polarization with 10 ng/mL LPS (E055:B55; Sigma-Aldrich) and 20 ng/mL IFNγ (PeproTech), (ii) for M2 polarization with 50 ng/ml human recombinant M-CSF, 20 ng/ml human recombinant IL-4, and 20 ng/ml human recombinant IL-13 (PeproTech). Human macrophages were then cultured alone or with 0.1ug, or 1ug SARS-CoV-2 Spike proteins for 48 hr. The cultured supernatants were collected, and cytokine levels determined using the bead-based immunoassays LEGENDplex™ Human Macrophage/ Microglia Panel (Biolegend). The assays were performed in 96-well plates following the manufacturer’s instructions. For measurements a FACSCelesta SORP flow cytometer (BD Biosciences) was employed, and data were evaluated with the LEGENDplex™ Data Analysis software.
Legendplex human macrophage microglia panel
Manufactured by BioLegend
Sourced in United States
The LEGENDplex Human Macrophage/Microglia Panel is a multiplex assay designed to measure the levels of specific analytes in human samples. It allows for the simultaneous quantification of multiple analytes related to macrophage and microglia functionality. The panel provides a comprehensive assessment of the target analytes without interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human il 4, by Thermo Fisher Scientific (2 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (2 mentions)
Recombinant human il 13, by Thermo Fisher Scientific (2 mentions)
Facs celesta sorp flow cytometer, by BD (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Lps e055 b55, by Merck Group (2 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Prism 8, by GraphPad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using legendplex human macrophage microglia panel
1
SARS-CoV-2 Spike Protein Modulates Macrophage Polarization
2
Measuring Macrophage Polarization in Sera and BFs
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M1 type (TNF-α, IFN-γ) and M2 type (arginase, TARC, IL-10, IL-1RA) macrophage marker concentrations in sera and BFs were determined using the bead-based immunoassays LEGENDplex Human Macrophage/Microglia Panel and Human Th Cytokines Panel (BioLegend, San Diego, CA, USA). The assays were performed in 96-well plates following the manufacturer's instructions. For measurements a LSR Fortessa flow cytometer (BD Biosciences) was employed, and data were evaluated with the LEGENDplex™ Data Analysis software.
3
Polarizing Macrophages for SARS-CoV-2 Spike Protein Interactions
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To polarize human macrophages, monocyte-derived macrophages were plated in 48-well plates at 5×104 cells per well in 500 µl RPMI medium and allowed to rest for 2 hr at 37°C before treating cytokines or LPS. The human M0 macrophages were either left untreated or treated for 48 hr to induce macrophage polarization: (i) for M1 polarization with 10 ng/ml LPS (E055:B55; Sigma-Aldrich) and 20 ng/ml IFNγ (PeproTech), (ii) for M2 polarization with 50 ng/ml human recombinant M-CSF, 20 ng/ml human recombinant IL-4, and 20 ng/ml human recombinant IL-13 (PeproTech). Human macrophages were then cultured alone or with 0.1 or 1 µg SARS-CoV-2 Spike proteins for 48 hr. The cultured supernatants were collected, and cytokine levels determined using the bead-based immunoassays LEGENDplex Human Macrophage/Microglia Panel (BioLegend). The assays were performed in 96-well plates following the manufacturer’s instructions. For measurements, a FACSCelesta SORP flow cytometer (BD Biosciences) was employed, and data were evaluated with the LEGENDplex Data Analysis software.
4
Quantifying αKG and Cytokines in moDCs
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The extracellular and intracellular levels of αKG were measured on day 2 and day 5 of moDCs cultures in cell-free culture supernatants and deproteinised cell lysates, by using the colourimetric and fluorometric alpha-KG assay kit, respectively, according to manufacturer's instructions (Abcam, Cambridge, UK). Prior to deproteinization, the total protein levels in cell lysates were determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer's instructions.
The supernatants from PHA-stimulated PBMC cultures were collected after 48h and assayed for cytokine measurements using the LEGENDplex™ Essential Immune Response panel (BioLegend). The supernatants from moDC cultures were assayed using LEGENDplex™ human Macrophage/Microglia panel (BioLegend), IL-27 and moDC/T cell co-cultures supernatants were analysed using specific sandwich enzyme-linked immunosorbent assay (ELISA) for IL-4, IFN-γ, IL-17, IL-10 and TGF-β (all from R&D Systems). All cytokine measurements were done in duplicates and the concentrations were calculated according to 5-parameter nonlinear fit curves (GraphPad Prism 8).
The supernatants from PHA-stimulated PBMC cultures were collected after 48h and assayed for cytokine measurements using the LEGENDplex™ Essential Immune Response panel (BioLegend). The supernatants from moDC cultures were assayed using LEGENDplex™ human Macrophage/Microglia panel (BioLegend), IL-27 and moDC/T cell co-cultures supernatants were analysed using specific sandwich enzyme-linked immunosorbent assay (ELISA) for IL-4, IFN-γ, IL-17, IL-10 and TGF-β (all from R&D Systems). All cytokine measurements were done in duplicates and the concentrations were calculated according to 5-parameter nonlinear fit curves (GraphPad Prism 8).
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