Journal: Bio-protocol

Article Title: Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease

doi: 10.21769/BioProtoc.5018

Figure Lengend Snippet: Antibody list for microglial phenotypes

Article Snippet: Disease-associated microglia (DAM) , CLEC7A , 9 μg/300 μL , APC , REA154 , Human , Miltenyi , 130102985 , 1:50.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immunoregulatory activity of the natural product laminarin varies widely as a result lt of its physical properties 1

doi: 10.4049/jimmunol.1701258

Figure Lengend Snippet: Saturation binding curves for rh and rm-Dectin-1 and two of the laminarins are shown. N=6 replicates for each laminarin and each receptor. At minimum a three log concentration range was employed with at least 10 data points for each analysis.

Article Snippet: Samples were analyzed for binding to recombinant human or mouse Dectin-1 (R&D Systems cat #1859-DC-050 and 1756-DC-050 respectively; both are C-terminal, extracellular truncated versions containing a 10 His N terminal tag) with an Octet Ks bio-layer interferometry instrument manufactured by ForteBio (Fremont CA).

Techniques: Binding Assay, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immunoregulatory activity of the natural product laminarin varies widely as a result lt of its physical properties 1

doi: 10.4049/jimmunol.1701258

Figure Lengend Snippet: A. Dectin-1 in RAW or THP-1 cells was silenced using lentiviral particles containing shRNA targeted against the murine and human Clec7a gene. Control cells were also lentivirally transduced with a non-targeting shRNA against GFP. After antibiotic selection, single cell clones were propagated and screened for loss of Dectin-1 mRNA expression using qPCR (left) or protein expression (right). For protein analysis in RAWs, cells were pretreated with 30 μg/ml zymosan. Numbers under blots represent relative level of Dectin-1 protein vs. control cells as determined by quantification using the software ImageJ and normalization to actin protein levels. Dashed lines indicate image splicing to remove irrelevant lanes. B. TNFα production in response to 24 h treatment of the indicated cells, control (red) or Dectin-1 knockdown (black; clone 6 and clone 3 for RAW and THP-1, respectively), with increasing concentrations of indicated laminarin. Graphs are one representative experiment of at least three replicates.

Article Snippet: Samples were analyzed for binding to recombinant human or mouse Dectin-1 (R&D Systems cat #1859-DC-050 and 1756-DC-050 respectively; both are C-terminal, extracellular truncated versions containing a 10 His N terminal tag) with an Octet Ks bio-layer interferometry instrument manufactured by ForteBio (Fremont CA).

Techniques: shRNA, Transduction, Selection, Clone Assay, Expressing, Software

Journal: The Journal of Immunology Author Choice

Article Title: Identification of novel protein biomarkers of macrophage polarization using comparative proteomic analyses of murine primary macrophages

doi: 10.1093/jimmun/vkaf202

Figure Lengend Snippet: Murine primary M0 macrophages were polarized into M1 or M2 macrophages. M0 BMDMs were incubated with LPS for 8 h to elicit M1 polarization or incubated with mouse IL-4 for 24 h to elicit M2 polarization. The cells were collected and subjected to polarization validation using qPCR (A and B), immunoblot (C), and flow cytometry (D). (A) Transcript levels of the well-established M1 biomarkers in M0, M1, and M2 macrophages. (B) Transcript levels of the well-established M2 biomarkers in M0, M1, and M2 macrophages. (C) Immunoblot analysis validated the unique expression of M1 biomarker Ccl5 in M1 cells and of M2 biomarker Clec7a in M2 macrophages. Gapdh served as a loading control. (D) Flow cytometry using antibodies against M1 biomarker iNOS and M2 biomarker Arg1 validated that the majority of LPS-treated macrophages were iNOS positive and IL-4-treated macrophages were mainly Arg1 positive. Data in the bar graphs are presented as mean ± SEM. n = 3–4. ** P < 0.01 relative to M0 BMDMs.

Article Snippet: Primary antibodies used in this study included antibodies against glyceraldehyde-3-phosphate dehydrogenase (Gapdh; Cell Signaling, Boston, MA, USA, 2118; 1:1,000); C-C motif chemokine ligand 5 (Ccl5; Proteintech, Rosemont, IL, USA, 12000-1-AP; 1:500); mouse C-type lectin domain family 4, member e (Clec4e; Novus Biologicals, Centennial, CO, USA, NBP2-27093SS; 1:250); human CLEC4E (Proteintech, 19376-1-AP; 1:1,000); C-type lectin domain family 7, member a (Clec7a; R&D Systems, Minneapolis, MN, USA, AF1756-SP; 1:2,000); mouse CD72 antigen (Cd72; R&D Systems, AF1279SP; 1:1,000); guanylate binding protein 2 (Gbp2; Proteintech, 11854-1-AP; 1:1,000); mouse aconitate decarboxylase 1 (Acod1; Cell Signaling, 19857T; 1:1,000); human ACOD1 (Cell Signaling, 77510T; 1:1,000); and neurochondrin (Ncdn; Proteintech, 13187-1-AP; 1:1000).

Techniques: Incubation, Biomarker Discovery, Western Blot, Flow Cytometry, Expressing, Control