Cd11c pacblue

Manufactured by BioLegend

CD11c is a cell surface glycoprotein expressed on the surface of dendritic cells, macrophages, and granulocytes. It is conjugated to the PacBlue fluorophore, which can be detected using flow cytometry.

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5 protocols using cd11c pacblue

Spinal Cord Injury Immune Cell Analysis

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Spinal cords isolated 1 week after injury and implantation were collected with the bridge and contralateral, but not rostral or caudal tissue, to limit myelin debris. Tissue was digested with 1 U mL⁻¹ liberase at 37°C for 6 minutes in thermomixer (Thermo Scientific) at 1400 RPM. Live cells were detected with a blue fix exclusion dye for 15 minutes at 4 °C. Cells were then incubated for an additional 30 minutes with Ly6G (PE, 1:1000, Biolegend), arginase (FITC, 1:1000, Abcam), CD4 (PECy7, 1:1000, Biolegend), F4/80 (Alexafluor 700, 1:1000, Biolegend), CD11c (PacBlue, 1:1000, Biolegend), GFAP (APC, 1:1000, BD), and CD45 (brilliant violet 510, 1:1000, Biolegend). Cells were then rinsed with saline, fixed with 4% paraformaldehyde for 10 minutes, and rinsed twice more. Samples were analyzed on a MoFlo Astrios flow cytometer using appropriate excitation lasers and emission filters (Beckman Coulter, Brea, CA, USA). Data was analyzed with FlowJo software (FlowJo, Ashland, OR, USA) by investigators blind to the condition.

Dumont C.M., Carlson M.A., Munsell M.K., Ciciriello A.J., Strnadova K., Park J., Cummings B.J., Anderson A.J, & Shea L.D. (2019). Aligned hydrogel tubes guide regeneration following spinal cord injury. Acta biomaterialia, 86, 312-322.

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Lung Single-Cell Immune Profiling by Flow Cytometry

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Lung single-cell suspensions [54 (link)] were stained with a fixable viability marker, stained with an antibody panel (below), and assayed on a FACS Canto II (BD; Franklin Lakes, NJ). For CD4 T cell population identification and determination of IL-33 cell sources, cells were stimulated for 5 hr with phorbol-12-myristate-13-acetate (PMA; 5 ng ml^-1) and ionomycin (500 ng ml^-1) in the presence of a protein transport inhibitor (GolgiPlug; BD) prior to intracellular staining with CD3—eFluor450, CD4—PerCP, IFNγ—PE, and IL-4—PE-Cy7 or CD45—PerCP-Cy5.5, EpCam—APC, CD31—PE-Cy7 (all eBioscience), and IL-33—PE (R&D Systems; Minneapolis, MN). For ILC2 staining, cells were labeled with antibodies to CD3—PacBlue, CD19—PacBlue, CD11c—PacBlue, CD11b—PacBlue, CD49b—PacBlue, F4/80—PacBlue, FcεRI—PacBlue, and Sca-1—PE-Cy7, (all BioLegend; San Diego, CA), ST2—FITC (MD Bioproducts; St. Paul, MN), Thy1.2 (CD90)—biotin (Southern Biotech; Birmingham, AL), Gata3—PE-Cy7 (BD), CD45—PerCP-Cy5.5, CD25—APC-Cy7/PE, and ICOS—APC (all eBioscience). Data were analyzed using FlowJo v10 (S1 Fig).

Saravia J., You D., Shrestha B., Jaligama S., Siefker D., Lee G.I., Harding J.N., Jones T.L., Rovnaghi C., Bagga B., DeVincenzo J.P, & Cormier S.A. (2015). Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33. PLoS Pathogens, 11(10), e1005217.

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Multicolor Flow Cytometry of Tumor Cells

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After blocking and live-dead staining, tumor-derived and cultured immune cells (BMMs, T cells and BMDCs) were stained with the following antibodies (all diluted 1:100) for 30 min at 4°C: rat anti-CD11b-BV711 (clone M1/70, BioLegend), rat anti-CD11b-PE-Cy7 (clone M1/70, eBioscience), rat anti-CD45-V500 (clone 30-F11, BD Biosciences), rat anti-CD45-PerCP-Cy5.5 (clone 30-F11, eBioscience), rat anti-F4/80-APC-Cy7 (clone BM8, BioLegend), rat anti-F4/80-AF647 (clone BM8, BioLegend), rat anti-Ly6G-BV605 (clone 1A8, BioLegend), rat anti-Ly6C-AF700 (clone HK1.4, BioLegend), CD3-APC (clone 17A2, BioLegend), CD8a-APC/Cy7 (clone 53-6.7, BioLegend), CD4-APC/eFluor780 (clone GK1.5, eBioscience), CD40-PE (clone: 3/23, BD Biosciences), CD86-PerCP-Cy5.5 (clone GL-1, BioLegend), CD80-FITC (clone 16-10A1, BioLegend), MHC II-AF647 (clone M5/114.15.2, BioLegend) and CD11c-PacBlue (clone N418, BioLegend). All samples were analyzed with an Attune NxT apparatus (Life Technologies). Compensation was performed using single-stained OneComp eBeads (Thermo Fisher Scientific).

Cianciaruso C., Beltraminelli T., Duval F., Nassiri S., Hamelin R., Mozes A., Gallart-Ayala H., Ceada Torres G., Torchia B., Ries C.H., Ivanisevic J, & De Palma M. (2019). Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. Cell Reports, 27(10), 3062-3080.e11.

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In Vivo Imaging of Dextran-Encapsulated cGAMP

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dextran was mixed at a 1:3 ratio of 70kDa Texas-red labeled dextran to 70 kDa dextran (ThermoFisher cat. D1830). The mixture of dextran was reacted with 2-ethoxypropene overnight. Mice were inoculated with 200,000 B16F10 tumor cells on day 0. On day 10, mice were treated with 10 μg of cGAMP encapsulated in Texas-red labeled Ace-DEX polymer. Texas-red Axe-DEX was made as described above with Texas-red labeled dextran obtained from Thermo Fisher (Waltham, MA cat. D1830). Twenty-four hours after treatment, mice were sacrificed the following organs were harvested: brain, lung, liver, kidney, spleen, lymph nodes, and tumor. The organs were then imaged using the IVIS Kinetic (PerkinElmer Waltham, MA). Samples were excited for 1 second at an excitation wavelength of 570 nm and detected using the Cy5.5 emission filter. The spleen was then taken and made into a single cell suspension. The splenocytes were stained (CD45 (BV-421), CD3 (PE-Cy7), CD4 (PerCP-cy5.5), CD8 (AF700), CD11b (APC), and CD11c (Pac-Blue); Biolegend, San Diego, CA), analyzed on a LSR II (BD Biosciences, San Jose, CA) and analyzed by FlowJo software (Tree Star, Ashland, OR).

Watkins-Schulz R., Tiet P., Gallovic M.D., Junkins R.D., Batty C., Bachelder E.M., Ainslie K.M, & Ting J.P. (2019). A microparticle platform for STING-targeted immunotherapy enhances natural killer cell and CD8+ T cell-mediated anti-tumor immunity.. Biomaterials, 205, 94-105.

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Flow Cytometric Analysis of Immune Cells

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D7-6-OHDA and saline-treated mice were perfused, and brains (ipsilateral and contralateral hemispheres) and spleens harvested and processed into single cell suspensions for flow cytometric analysis. Briefly, brain and spleen tissues were mechanically homogenized in CNS (2.5% HEPES pH 7.5, Invitrogen in Hanks' Balance Salt Solution (HBSS) without Ca/Mg, Gibco) and FACS buffer (2% Fetal Bovine Serum in PBS), respectively. Myelin was removed from brain samples using a percoll gradient (25% percoll in CNS buffer). Resulting single cell suspensions were stained for live/dead (aqua, ThermoFisher Scientific), TREM1 (APC, R&D Systems), CD11c (PAC-Blue, Biolegend), Ly-6G (PE-Cy7, Biolegend), CD11b (APC-Cy7, Biolegend), CD3 (PE, Biolegend), and CD45 (PerCP-Cy5.5, Biolegend) to isolate immune cell populations. Cells were fixed in 2% PFA (Santa Cruz Biotechnology) prior to analysis. Data was gated (Supplemental Figure 1) analyzed using FlowJo (version 10.7.1).

Lucot K.L., Stevens M.Y., Bonham T.A., Azevedo E.C., Chaney A.M., Webber E.D., Jain P., Klockow J.L., Jackson I.M., Carlson M.L., Graves E.E., Montine T.J, & James M.L. (2022). Tracking Innate Immune Activation in a Mouse Model of Parkinson's Disease Using TREM1 and TSPO PET Tracers. Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 63(10).

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