Journal: eLife

Article Title: IRF4 haploinsufficiency in a family with Whipple’s disease

doi: 10.7554/eLife.32340

Figure Lengend Snippet: CD11b, CD86, CD206, CD209 and HLA-DR mean fluorescence intensity (MFI) for M2-MDM (left) and M1-MDM (right) from P1 and two healthy unrelated controls ( C1 and C2 ), either left non-stimulated (NS) or stimulated with IL-4 (for M2-like MDMs) or IFN-g (for M1-like MDMs). We showed that CD11b, CD86, CD206, CD209, and HLA-DR expression levels were similar in MDMs from P1 and healthy unrelated controls.

Article Snippet: Antibody , anti-CD206 , Miltenyi Biotec , #130-099-732 , Fluorochrome: PE.

Techniques: Fluorescence, Expressing

Journal: eLife

Article Title: IRF4 haploinsufficiency in a family with Whipple’s disease

doi: 10.7554/eLife.32340

Figure Lengend Snippet:

Article Snippet: Antibody , anti-CD206 , Miltenyi Biotec , #130-099-732 , Fluorochrome: PE.

Techniques: Plasmid Preparation, Isolation, Purification, Transfection, Construct, Generated, Recombinant, Sequencing, TA Cloning, Expressing, Mutagenesis, Staining, Migration, Software

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1 and TNF- α protein expression in 33.3 mM glucose-induced macrophage. (a) iNOS and CD206 protein expressions. (d) Arg-1 protein expression. (f) TNF- α protein expression. The results of iNOS, CD206, Arg-1, and TNF- α were represented in (b), (c), (e), and (g), respectively. All results were expressed as a ration with respect to control and represented as the mean ± SD in triplicates. ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing, Control

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1, and TNF- α mRNA expressions in 33.3 mM glucose-induced macrophage. (a) iNOS mRNA expression. (b) CD206 mRNA expression. (c) CD206 mRNA expression. (d) TNF- α mRNA expression.All the results were represented as the mean ± SD in triplicates ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Neuropeptide Y Promotes Human M2 Macrophage Polarization and Enhances p62/SQSTM1-Dependent Autophagy and NRF2 Activation

doi: 10.3390/ijms232113009

Figure Lengend Snippet: Flow cytometric analysis of the M1 markers HLA-DR and CD16 and the M2 markers CD206 and CD163 on ( a ) M0, ( b ) polarized M(IFN-γ/LPS), and ( c ) polarized M(IL-10) macrophages. Macrophages (7 × 10 5 cells per mL) were stimulated or not with 10 −8 M NPY in the presence or absence of LPS in complete medium for 24 h and then analyzed for surface marker expressions by flow cytometry. Histograms show the percentages of positive cells (%) and the mean fluorescence intensity (MFI). Results are expressed as mean value ± SD of 4 independent experiments. Significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis; *: p < 0.05, **: p < 0.01.

Article Snippet: To determine macrophage phenotypic surface markers, macrophages were stained with the following monoclonal antibodies (mAbs): phycoerythrin (PE)-CD163, fluorescein isothiocyanate (FITC)-CD206 and PE-Vio770-CD206 mAbs (Miltenyi Biotec), allophycocyanin (APC)-CD16 and APC-Alexa Fluor 750-HLA-DR (clone Immu357) mAbs (Beckman Coulter, Lane Cove, Australia), PE-CD1a and FITC-CD14 mAbs, or with isotype-matched control mAbs from PharMingen (PharMingen, San Diego, CA, USA) for 30 min at 4 °C.

Techniques: Marker, Flow Cytometry, Fluorescence

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables identification of all hepatic cell types including bona fide cell doublets, related to Figure 1 (A and B) Top DEGs (A) and DEPs (B) for cell types from Figure 1 B. (C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocols; 71,162 cells from ex vivo digestions, 96,066 cells from in vivo digestions, and 18,666 nuclei. Numbers on plots represent numbers of cells/nuclei per population. (D) Correlation plots showing genes captured within the KC, B cell and neutrophil populations with and without addition of CITE-seq antibodies. (E) Expression of VSIG4, CD206, and ESAM (protein, top) and Vsig4 , Mrc1 , and Esam (mRNA, bottom). (F) UMAP showing clusters of cells when only minimal QC for gene number and % mitochondrial genes is performed; 17,669 cells pooled from 3 samples. Expression of Cd5l, Cd19 , and Kdr by the clusters facilitating identification of cell types per annotation. (G) CITE-seq data from (F) in Flow-Jo showing expression of CD206 and ESAM in total KCs (left) and total B cells (middle). Numbers represent % of entire KC or B cell population. Identified populations were then mapped back onto the original UMAP (right). (H) Expression of CD31, CD26, and CD38 by indicated populations. (I) Heatmaps showing expression of top DEGs between KC1s and LSECs (left), KC2s and KC1s + LSECs (middle) and B cell2s and B cell1s + LSECs (right). (J) 3D reconstruction of murine liver following perfusion with antigen fix to inflate endothelial cells and staining with antibodies against CD31, CD206, and F4/80. (K) UMAP showing clusters generated from Visium analysis of liver tissue (4 samples) and liver capsule (1 sample). (L) Top unbiased genes defining zonation trajectory from portal to central vein in Visium. (M) Expression of Glul and Epcam by confocal microscopy (left), annotation of portal, periportal, mid, and central regions on same tissue section (middle) and overlay of both datasets (right). (N) Identification of cholangiocyte (left) and cDC (right) signatures on zonated Visium spots. (P) Molecular Cartography showing expression of indicated zonated hepatocyte mRNAs in liver tissue. Data are representative of 2 mice. (O) Expression of Itgae (encoding CD103) in the UMAP of the total liver (left) and flow cytometric analysis of total cDC1s for CD103 and MHCII expression in the healthy murine liver (right).

Article Snippet: Anti-Human CD206 (REAL518) APC , Miltenyi Biotec , 130-122-168; RRID: AB_2857557.

Techniques: Isolation, Ex Vivo, In Vivo, Expressing, Staining, Generated, Confocal Microscopy

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet:

Article Snippet: Anti-Human CD206 (REAL518) APC , Miltenyi Biotec , 130-122-168; RRID: AB_2857557.

Techniques: Purification, Recombinant, Staining, cDNA Synthesis, Gene Expression, Software, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung

doi: 10.1084/jem.20232192

Figure Lengend Snippet: SARS-CoV-2 entry, replication, and productive infection of purified AMs and IMs. (a) Strategy for purification, culture, and infection of human lung macrophages with SARS-CoV-2 virions or a SARS-CoV-2 Spike-pseudotyped lentivirus. Human lung tissue obtained from surgical resections or organ donors was dissociated fresh, then enriched for macrophages by MACS using antibodies against the general lung macrophage antigen CD206, followed by specific AM or IM purification using FACS for distinguishing markers . Purified AMs (CD206 + CD204 hi ) or IMs (CD206 + CD204 lo ) were cultured at 37°C in DMEM/F12 medium with 10% FBS, and either infected with SARS-CoV-2 virions and analyzed as indicated (Cases 11–12, panels b–f), or tested for viral entry and the effect of inhibitors and mABs using a SARS-CoV-2 Spike-pseudotyped lentivirus lenti-S-NLuc-tdT that encodes an NLuc reporter (Cases 6–10, 5 bio-replicates, panels g and h). For SARS-CoV-2 infections, purified AMs or IMs were mock-infected or exposed for 2 h to untreated or UV-inactivated (UVi) SARS-CoV-2 virions (MOI 0.05 or 0.01), washed to remove free virions, and infection continued for 48 h before assaying supernatant for virion production by plaque assay or analyzing the infected cells by fluorescence in situ hybridization (FISH using HCR) and IF staining (one bio-replicate shown). (b and c) Infection intermediates and morphologies of SARS-CoV-2–infected AMs (b) or IMs (c) generated as above and then fixed and IF-labeled for lysosomal antigen LAMP1 (green) and the infection dsRNA intermediate (red), followed by HCR for viral genomic RNA (light blue) and DAPI nuclear counterstain (dark blue). Examples of the observed infection classes are shown and their features summarized at panel bottom: Class 0 (non-infected), no expression of either dsRNA or viral gRNA; Class I (early infection): expression of dsRNA only; Class II (intermediate infection): co-expression of dsRNA and viral gRNA; Class III (advanced infection): expression of viral gRNA only; Class IV (aggregates): expression of globular viral gRNA bodies; Class V (cell destruction/death): weak or non-staining of DAPI nuclear stain and LAMP1, and expression of viral RNA. Scale bars, 1 µm. (d) Quantification of panels b and c showing the relative abundance of each infection class. Values above each bar, number of cells scored per condition. (e) SR microscopy of viral gRNA for Class 0/I AM, Class II/III IM, and Class IV IM from panels b and c. Note the large, globular viral gRNA aggregates (“RNA bodies”) throughout the cytoplasm in the class IV IM. Scale bars, 2 µm. (f) SARS-CoV-2 virions released into the medium by the above infected AMs or IMs, as determined by plaque assay of the indicated culture supernatants on a monolayer of VeroE6 cells. pfu, plaque-forming units. (g) Viral entry into AMs and IMs depends on SARS-CoV-2 Spike. Left: Diagram of lenti-S-NLuc-tdT, a lentivirus pseudotyped to express full length SARS-CoV-2 Spike, encoding both S1 and S2 subunits from the D614G variant (Spike+, D614G) protein on its surface and also engineered to express the reporter gene (boxed) encoding nuclear-targeted nanoluciferase (H2B- NLuc) and tdTomato fluorescent protein, separated by a self-cleaving T2A peptide. Right: Lenti-S-NLuc-tdT (Spike+ [D614G]) or a non-pseudotyped control lentivirus (Spike−) were added to purified AMs or IMs (Cases 11–12, two bio-replicates) in culture, and, after 4 h, free virions were washed off and infections continued for 48 h before quantification of infection by expression level (luminescence) of the NLuc reporter. Uninfected AMs or IMs (cells only) served as background control. RLU, relative light units. NLuc luciferase values are presented as mean ± SD from two independent experiments, with values normalized to control (non-neutralized) viral infections in each plate. Statistical test used was the unpaired t test. P values are computed by comparing Spike+(D614G) to Spike-controls. ***, P < 0.001. (h) Neutralization of lenti-S-NLuc-tdT entry into purified AMs (left) or IMs (right) from Cases 6–10 (five bio-replicates) by the indicated blocking antibodies against ACE2, CD169, or CD209.

Article Snippet: The isolated lung cells were stained with CD206 antibody conjugated to biotin (130-095-214; Miltenyi), washed twice with MACS buffer, then stained with Anti-Biotin MicroBeads (130-090-485; Miltenyi) and passed through an LS MACS column on a MidiMACS Separator magnet or a SuperMACS II Separator magnet, with the retained population designated “MACS CD206 + ” and further purified by fluorescence-activated cell sorting (FACS; see below).

Techniques: Infection, Purification, Cell Culture, Plaque Assay, Fluorescence, In Situ Hybridization, Staining, Generated, Labeling, Expressing, Microscopy, Variant Assay, Control, Luciferase, Neutralization, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung

doi: 10.1084/jem.20232192

Figure Lengend Snippet: FACS and scRNA-seq characterization of purified AMs and IMs. (a–e) FACS gating and scRNA-seq or purified AMs and IMs from Case 11 (one bio-replicate shown). (a) Sequential FACS data and sorting gates (red) for dissociated human lung cells, following MACS enrichment of lung resident macrophage (MACS CD206 + ) cells. Cells were first gated on viable single cells that were CD45 + and CD206 + (left panel), then two gates were subsequently sorted (right panel) for 10x scRNA-seq transcriptomic profiling: CD206 + CD204 hi (putative AMs), and CD206 + CD204 lo (putative IMs). (b) Flow cytometry of the sorted AMs and IMs, with flow gating defined as in . Results are shown for staining of various surface antigens reported to distinguish AMs and IMs, including CD14 (upper left), CD16 (center top), HLA-DR (left bottom), CD11b (center bottom), and CD11c (lower right), as well as for staining of the canonical SARS-CoV-2 receptor ACE2 (upper right). Note that although neither AMs nor IMs express ACE2 mRNA , AMs, but not IMs, express ACE2 protein. (c) UMAP projection of sorted putative AMs and putative IMs from (a), with the transcriptomic molecular cell annotations indicated, including AMs, IMs, proliferating macrophages, and rare mast/basophils. (d) The same UMAP projection colored by sorting gate metadata. Note the correspondence between the scRNA-seq molecular annotation and the gating metadata. (e) The relative frequencies of the molecular types of AMs and IMs in each of the indicated sorting gates; in the CD206 + CD204 hi channel, AMs were 88%, IMs were 1%, and proliferating macrophages were 11%; in the CD206 + CD204 lo channel, IMs were 81%, AMs were 17%, proliferating macrophages were 1%, and mast/basophils were 1%.

Article Snippet: The isolated lung cells were stained with CD206 antibody conjugated to biotin (130-095-214; Miltenyi), washed twice with MACS buffer, then stained with Anti-Biotin MicroBeads (130-090-485; Miltenyi) and passed through an LS MACS column on a MidiMACS Separator magnet or a SuperMACS II Separator magnet, with the retained population designated “MACS CD206 + ” and further purified by fluorescence-activated cell sorting (FACS; see below).

Techniques: Purification, Flow Cytometry, Staining

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Photobiomodulation mitigates chondrocyte catabolism in osteoarthritis by modulating macrophage M1 polarization through the IL-6/JAK/STAT pathway

doi: 10.1186/s13018-025-06506-4

Figure Lengend Snippet: The IF staining and the representative micro-CT images of mouse knees. A The effects of CLD Lips and PBM intervention on macrophage depletion by IF staining ( n = 4). F4/80 is a pan-macrophage marker (green). B The effects of CLD Lips and PBM intervention on macrophage polarization by IF staining ( n = 4). CD86 is a M1 macrophage marker (green), and CD206 is a M2 macrophage marker (red). C 2D reconstruction of micro-CT images of knee joints. D BV/TV and E Tb.Sp of micro-CT analysis ( n = 5). (Data are presented as mean ± SD; Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: After cooling and blocking, sections were incubated overnight at 4 °C with the second primary antibody, anti-CD206 antibody (Cat. No. A02285-2, rabbit polyclonal, Bosterbio, USA) diluted 1:100.

Techniques: Staining, Micro-CT, Marker