Cd11b alexafluor700

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD11b-AlexaFluor700 is a fluorescently-labeled antibody that binds to the CD11b cell surface antigen. It is commonly used in flow cytometry applications to identify and quantify myeloid cells, such as monocytes and granulocytes, in biological samples.

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6 protocols using cd11b alexafluor700

Macrophage migration into mouse adipose tissue

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In vivo macrophage migration into epididymal fat pads of mice was performed as published earlier 4, 23. Ten‐week‐old male C57BL/6 wild‐type (WT) mice purchased from Charles River Laboratory (Wilmington, DE, USA) were used. All mice were fed with standard chow and water ad libitum and experiments were approved by Yale University's Institutional Animal Care and Use Committee (2015‐10756). Five mg/kg LPS (Sigma‐Aldrich, St. Louis, MO, USA) only, LPS together with either 20 μg mouse D‐DT or the equal amount of mouse MIF was injected into the epididymal fat pads via a small incision to induce local adipose tissue inflammation. Control mice were treated with the equal volume of PBS. Thioglycollate elicited peritoneal macrophages (PM) were labelled with Cell Tracker Green CMFDA (Cell Tracker Green CMFDA Dye; Life Technologies, Carlsbad, CA, USA). Next, labelled PMs were injected retro‐orbitally. Mice were killed after 48 hrs and epididymal fat tissue was harvested, and the stromal vascular fraction (SVF) was isolated. The isolated ATM were stained with Cd11b AlexaFluor700 (eBioscience, San Diego, CA, USA), F4/80 eFluor450 (eBioscience) and Cd45 APC (Biolegend, San Diego, CA, USA) as reported earlier 4.

Kim B., Tilstam P.V., Hwang S.S., Simons D., Schulte W., Leng L., Sauler M., Ganse B., Averdunk L., Kopp R., Stoppe C., Bernhagen J., Pallua N, & Bucala R. (2016). D‐dopachrome tautomerase in adipose tissue inflammation and wound repair. Journal of Cellular and Molecular Medicine, 21(1), 35-45.

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Quantifying FITC-positive Macrophages in Adipose Tissue

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The content of FITC-positive macrophages in the harvested adipose tissue was measured by flow cytometry as described previously [16 (link)]. In short, adipose tissue was minced and stromal vascular fraction (SVF) cells yielded by digestion with Collagenase Type II (Sigma Aldrich, St. Louis, United States). Following antibodies were used for surface staining: CD11b-AlexaFluor700 (eBioscience, San Diego, United States), F4/80-eFluor450 (eBioscience, San Diego, United States), CD45-APC (Biolegend, San Diego, United States). Analyses were performed on a LSR II cytometer (BD Bioscience, San Jose, United States).

Kim B.S., Rongisch R., Hager S., Grieb G., Nourbakhsh M., Rennekampff H.O., Bucala R., Bernhagen J, & Pallua N. (2015). Macrophage Migration Inhibitory Factor in Acute Adipose Tissue Inflammation. PLoS ONE, 10(9), e0137366.

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Murine Monocyte Subpopulations Identification

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Peripheral blood (∼100 μL) was obtained via facial vein puncture and collected into BD Microtainer EDTA tubes (BD Biosciences). Erythrocytes were lysed with 2 mL of RBC lysis buffer (eBiosciences) for 5 min on ice, followed by quenching with 10 mL of cold PBS. Leukocytes were collected by centrifugation (380g for 10 min at 4 °C) and resuspended in ice-cold flow cytometry staining buffer (eBioscience). Cell suspensions were incubated for 30 min on ice with fluorophore-labeled cell surface antibodies CD45-605NC, F4/80-eFluor 450, CD11b-Alexa Fluor 700 (eBioscience), and Ly6C-FITC (BD Biosciences). After staining, cells were centrifuged and resuspended in PBS and analyzed on a BD LSRII flow cytometer. Non-debris gates were established on SSC/FSC plots and surface marker positivity was determined from histograms and dot plots. Isotype control samples were used to determine negative fluorescence thresholds. From the lymphocytemonocyte gate, pro-inflammatory (CD45⁺CD11b⁺F4/ 80^lowLy6C^hi) and patrolling (CD45⁺CD11b⁺F4/80^low-Ly6C^low) monocytes were identified [19 (link), 26 (link), 44 (link)]. Final analysis was performed using FlowJo v.7.6 software.

Kingery J.R., Hamid T., Lewis R.K., Ismahil M.A., Bansal S.S., Rokosh G., Townes T.M., Ildstad S.T., Jones S.P, & Prabhu S.D. (2017). Leukocyte iNOS is required for inflammation and pathological remodeling in ischemic heart failure. Basic research in cardiology, 112(2), 19.

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Multiparametric Flow Cytometry Immunoprofiling

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The following anti-mouse mAbs were used for surface staining during flow cytometry: CD3-eFluor 450, CD8-PerCP-Cy5.5, CD11b-AlexaFluor 700, CD11b-eFluor 450, CD14-FITC, CD25-APC, CD25-eFluor 450, CD45.1-APC, Ly6C-APC-eFluor 780, I-A/E-FITC (eBioscience, San Diego, CA, USA), CD45.1-eFluor 450, CD45R-Horizont V500, CD3-Horizont V500, CD4-Horizont V500, CD4-PerCP, CD8-Horizont V500, Ly6G-AlexaFluor 700, Siglec F-PE (BD Biosciences, San Jose, California, USA) and TLR4-APC (BioLegend, San Diego, Ca, USA). For intracellular staining, the following anti-mouse mAbs were used: TNFα-PE, Foxp3- and IFNγ-PE (eBioscience; San Diego, California, USA). Fc-block (anti-CD16/CD32 mAbs; eBioscience, San Diego, California, USA) was used both in surface and intracellular staining. For ELISA, matched anti-mouse capture mAb and biotinylated detection mAb against TNF-α, IFN-γ, IL-1β, IL-12 and IL-6 were used (R&D System, Minneapolis, Minnesota, USA). Blocking anti-mouse CD25 (PC61.5), CD4 (GK1.5) and anti-IFN-γ (XMG1.2) mAbs, as well as anti-mIL-2 mAbs for preparing IL-2 complexes (S4B6, JES6-1A12), were obtained from BioXcell (Lebanon, New Hampshire, USA).

Tomala J., Weberova P., Tomalova B., Jiraskova Zakostelska Z., Sivak L., Kovarova J, & Kovar M. (2021). IL-2/JES6-1 mAb complexes dramatically increase sensitivity to LPS through IFN-γ production by CD25+Foxp3- T cells. eLife, 10, e62432.

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Immune Cell Profiling in Murine Bone Marrow, Spleen, and Lymph Nodes

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Immune cell populations in the bone marrow, spleen, and inguinal lymph nodes of female mice 8 weeks after OVX or sham surgery were determined via flow cytometric analysis. Inguinal lymph nodes and spleen were explanted and passed through a cell strainer (70 µm). The ends of the femur were removed, and bone marrow was centrifuged out (12,300 rpm for 40 s) and resuspended in phosphate-buffered saline. Lysis of erythrocytes of spleen and bone marrow samples was performed by using lysis buffer (150 nM NH₄CL, 1 mM KHCO₃, 0.1 mM Na₂EDTA, all Sigma-Aldrich) for 5 min at 37 °C. Cells were stained with the following antibodies for 30 min on ice: F4/80 FITC (1:50, eBioscience, Frankfurt, Germany), Ly6G V450 (1:400, BD Bioscience, Heidelberg, Germany), CD11b Alexa Fluor 700 (1:400), CD3e PE-Xyanine7 (1:100), CD4 APC-eFluor 780 (1:200), CD8a APC (1:800), and CD19 PE (1:400, all eBioscience). Corresponding isotype controls were used. Live–dead discrimination was performed with 7-aminoactinmycin D. A BD FACSLyric^™ flow cytometer (BD Bioscience) was used for sample measurement, and FlowJo software (10.0.8r1, FlowJo, Ashland, OR, USA) was used for analysis.

Fischer V., Bülow J.M., Krüger B.T., Ragipoglu D., Vikman A., Haffner-Luntzer M., Katsoulis-Dimitriou K., Dudeck A, & Ignatius A. (2023). Role of Mast-Cell-Derived RANKL in Ovariectomy-Induced Bone Loss in Mice. International Journal of Molecular Sciences, 24(11), 9135.

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Isolation and Characterization of Peritoneal Macrophages

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Peritoneal macrophages (PMs) and intraperitoneal fluid were collected from euthanized mice by peritoneal lavage with 10 ml PBS and centrifuged at 300 g for 5 minutes. The supernatants were collected for protein measurement and stored at −20 °C. The remaining cell pellet containing the PMs was treated with ammonium-chloride-potassium (ACK) lysis buffer (Lonza, NJ, USA) for erythrocyte lysis. The cells then were washed, centrifuged, and counted in a Countess® II Automated Cell Counter (Thermo Fisher Scientific, MA, USA) with dead cells excluded by trypan blue, and then used for further experiments. PMs were characterized in two separate subsets by flow cytometric analysis. Briefly, cells were labeled at 4 °C for 30 min in FACS Buffer with the following fluorophore-conjugated antibodies: CD11b-AlexaFluor700 (eBioscience, CA, United States), F4/80-eFluor450 (eBioscience, CA, United States), CD45-APC-Cy7 (BD Pharmingen, CA, United States). Flow cytometry was performed on a LSR II cytometer (Becton Dickinson, Franklin, NJ) and analyzed with Flow Jo software (Tree Star, Ashland, OR). We characterized the different PM subsets as F4/80^high, CD11b⁺ or F4/80^low, CD11b⁺.

Kim B.S., Tilstam P.V., Arnke K., Leng L., Ruhl T., Piecychna M., Schulte W., Sauler M., Frueh F.S., Storti G., Lindenblatt N., Giovanoli P., Pallua N., Bernhagen J, & Bucala R. (2020). Differential regulation of macrophage activation by the MIF cytokine superfamily members MIF and MIF-2 in adipose tissue during endotoxemia. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(3), 4219-4233.

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