Streptavidin apc cy7

Manufactured by BD

Sourced in United States

Streptavidin-APC-Cy7 is a fluorescent conjugate used in flow cytometry and other immunoassays. It consists of the protein streptavidin labeled with the fluorescent dyes allophycocyanin (APC) and Cyanine 7 (Cy7).

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35 protocols using streptavidin apc cy7

Comprehensive Antibody Panel for Cell Profiling

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The following primary antibodies were used for experiments: CD45-PE Cy7, CD45-APC Cy7, and CD45 APC (clone 30-F11, BD Biosciences, BioLegend), pan-keratin (Polyclonal, Dako), EpCAM-PE (clone G8.8, eBioscience), FoxN1 (clone G-20, Santa Cruz Biotechnology), FSP1 (clone S100A4, BioLegend), Thy1.2 (clone 30-H12, BD Biosciences), CD11b (clone, BioLegend), CD11b PerCpcy5.5 (clone M1/70, BioLegend), CD4-biotinylated (clone RM4-5, BioLegend), CD8-biotinylated (clone 53-6.7, BioLegend), CD19 (clone 1D3, BioLegend), rabbit anti-GFP (Life), and CD205 (LY75/DEC-205) (clone HD30 (Millipore). We also used lineage depletion panel containing B220. As control for EpCAM staining, we used rat IgG2a, κ isotype control (eBioscience). As control for CD45 staining, we used rat IgG2b, κ isotype control (BioLegend).
The following secondary reagents were used for experiments: donkey anti-rabbit IgG-TRITC, donkey anti-rabbit IgG-Cy5, donkey anti-rabbit IgG-FITC, donkey anti-rat IgG-TRITC, donkey anti-goat IgG-FITC, and goat anti-rat IgM-TRITC (Jackson ImmunoResearch); anti-rat IgG2a-FITC, anti-rat IgM-FITC, streptavidin-APC, streptavidin-APC Cy7, and streptavidin-PerCP Cy5.5 (BD Biosciences); and streptavidin-TRITC (Southern Biotechnology Associates).

Chakrabarti S., Hoque M., Jamil N.Z., Singh V.J., Pollacksmith D., Meer N, & Pezzano M.T. (2022). Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs. Journal of Immunology Research, 2022, 6061746.

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Characterization of Murine Kidney Cell Populations

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Medaka mesonephros and adult C57Bl/6 mouse kidneys were mechanically dissociated to a single cell suspension. Whole kidneys and KKPS5 cells were stained for CD133-PE, CD34-PE, CD105-PE, CD90.2-FITC (eBiosciences, Inc., San Diego, CA), Sca-I–Pacific Blue, c-kit-PE-Cy7 (BioLegend, San Diego, CA), CD31-FITC, CD24-FITC, CD106-FITC, FLT-3-PE, CD9-Biotin, and Streptavidin-APC-Cy7 (BD Biosciences), and analyzed by flow cytometry using an LSRII (BD Biosciences) with FACSDiva (BD Biosciences) software version 4.1. Appropriate isotype controls (BD Biosciences, eBiosciences, Inc., BioLegend) were used for analyses. Doublet exclusion was used to ensure analyses of single cells prior to forward/side scatter gating. Data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR) software version 10.

Webb C.F., Ratliff M.L., Powell R., Wirsig-Wiechmann C.R., Lakiza O, & Obara T. (2015). A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures. Biochemical and biophysical research communications, 463(4), 1334-1340.

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Multicolor Flow Cytometry Analysis

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Cocultured cells were analyzed by staining with CD45.1 PE, CD45.2 PE, c-Kit APC (eBioscience, San Diego, CA), Sca-1 PE-Cy7, streptavidin APC-Cy7 (BD Pharmingen) and lineage cocktail biotin (a component of StemSep, STEMCELL Technologies). Labeled cells were analyzed using an LSRII flow cytometer (BD Biosciences).

Jeong S.Y., Lyu J., Kim J.A, & Oh I.H. (2020). Ryk modulates the niche activity of mesenchymal stromal cells by fine-tuning canonical Wnt signaling. Experimental & Molecular Medicine, 52(7), 1140-1151.

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Phenotypic Characterization of Murine Splenocytes

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Mice were immunized i.p. with 25 μg/animal of CpG ODN 1826 (InvivoGen) or with 5 μg/animal of Salmonella flagellin (FliC). 6 h after immunization, mice were euthanized and splenocytes were labeled. Fc receptors were blocked with Fc Block (BD Biosciences) and subsequently stained first with anti-CD19-Biotin (clone 1D3), anti-CD3-Biotin (clone 145.2C11), and anti-CD49b-Biotin (clone DX5) for 40 min on ice. After two washes with PBS-2% FBS, cells were then incubated anti-MHCII (I-A/I-E)-Alexa Fluor 700 (clone M5/114.15.2), anti-CD11c-BV421 (clone N418), anti-CD11b-PE.Cy7 (clone M1/70), anti-CD8α-BV786 (clone 52–67), anti-CD80-FITC (clone 16-10A1), anti-CD86-APC (clone GL1), anti-CD40-PE (clone 1C10), Streptavidin APC.Cy7 (all antibodies and the streptavidin were purchased from BD Biosciences) and Live and Dead Aqua (Life Technologies). Flow cytometry was performed using LSRFortessa (BD Biosciences) and results were analyzed in FlowJo software (version 9.3, Tree Star, San Carlo, CA, USA).

Antonialli R., Sulczewski F.B., Amorim K.N., Almeida B.D., Ferreira N.S., Yamamoto M.M., Soares I.S., Ferreira L.C., Rosa D.S, & Boscardin S.B. (2017). CpG Oligodeoxinucleotides and Flagellin Modulate the Immune Response to Antigens Targeted to CD8α+ and CD8α− Conventional Dendritic Cell Subsets. Frontiers in Immunology, 8, 1727.

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Immunophenotyping of Immune Cell Subsets

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Single-cell suspensions of BM, spleen, and liver were prepared [14 (link), 15 (link)]. Antibody staining of cells was performed at 4 °C for 30 min. For biotinylated primary antibodies, the washed cells were further stained with fluorochrome-conjugated streptavidin or secondary antibodies. Cells were washed in phosphate-buffered saline-bovine serum albumin (PBS-BSA) buffer and subjected to either analysis or sorting (FACS AriaIII; BD Pharmingen, San Diego, CA, USA). The antibodies and conjugates used for the study were anti-CD4/biotin, anti-CD25/PE, anti-Foxp3/AF647, Streptavidin/PerCP, and Streptavidin/APCCy7 (all from BD Pharmingen); anti-CD11c/PE and anti-CD44/eFluor 450 (both from eBioscience, San Diego, CA, USA); and anti-CD31/biotin (BioLegend, San Diego, CA, USA).

Kochat V., Kanjirakkuzhiyil S., Baligar P., Nagarajan P, & Mukhopadhyay A. (2015). Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells. Stem Cell Research & Therapy, 6(1), 129.

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Multi-Marker Flow Cytometry Analysis

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The following antibodies and reagents were used for sample analysis by flow cytometry: Human Fc receptor binding inhibitor (eBioscience), Anti-CD45 PE-Cy7 (HI30; eBioscience), anti-CD11b PE (M1/70; BD Biosciences), anti-CD56 BV421 (HCD56; Biolegend), anti-CD3 PerCp-Cy5.5 (OKT3, eBioscience), anti-CD4 Alexa Fluor 700 (OKT4; eBioscience), anti-CD8a APC-Cy7 (HIT8a; Biolegend), anti-Vα24 PE (C15; Beckman Coulter), anti-Vβ11 FITC (C21; Beckman Coulter), anti-BDCA-2 APC (201A; Biolegend), anti-TCRγδ PE-CF594 (B1; BD Biosciences), anti-CD11c BV421 (3.9; Biolegend), anti-HLA-DR PE-CF594 (G46-6; BD Biosciences), anti-Cytokeratin 10 biotin (DE-K10; Abcam), anti-Cytokeratin 14 FITC (LL002; Abcam), Streptavidin-APC-Cy7 (BD Biosciences), and the anti-Human FoxP3 Staining Set (236A/E7; eBioscience). Rat IgG2a,κ Isotype Control APC (R35-95; BD Biosciences) was used as an isotype control for intracellular FoxP3 staining. Dead cells were excluded using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, New York, U.S.) according to the manufacturers’ instructions, and anti-CD45 was used to differential immune cells from non-immune cells.

Freeman A., Bridge J.A., Maruthayanar P., Overgaard N.H., Jung J.W., Simpson F., Prow T.W., Soyer H.P., Frazer I.H., Freeman M, & Wells J.W. (2014). Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage. PLoS ONE, 9(10), e110928.

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Multiparametric Flow Cytometry of Hematopoietic Cells

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Cells were stained for 20–30 min at 4 °C in the dark with directly labeled human monoclonal antibodies directed against CD11b-, CD11c-, Gr1-, CD3-, CD4-, CD8a-, CD19-, B220-, NK1.1, and TER119-conjugated to biotin, cKit (CD117)-APC, Sca1-PECy7, and streptavidin APC Cy7 (BD Biosciences and eBiosciences, USA). Cells were washed and then resuspended with PBS supplemented with 2% FBS and 2 mM Ethylenediaminetetraacetic acid (EDTA; Gibco, USA). Flow cytometric analysis was performed using FACS LSRII (BD Biosciences, USA) and FlowJo Software (USA).
Cell cycling was assessed with carboxyfluorescein (CFSE) incorporation at the start of the co-culture. F0 peaks were determined using the control sample of BMCs flowed through the micro-reactor without stromal support.

Khong D., Li M., Singleton A., Chin L.Y, & Parekkadan B. (2018). Stromalized microreactor supports murine hematopoietic progenitor enrichment. Biomedical microdevices, 20(1), 13.

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Immunophenotyping of Lymphoma and TAMs

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Lymphoma immunophenotype was assessed as previously described [22 (link)]. The following antibodies were used for staining: CD19-APC, B220 (CD45R)-FITC, IgM biotin, IgD-PE, and Streptavidin-APC/Cy7 (BD Biosciences, USA). Data were collected by a flow cytometer (BriCyteE6, Mindray Bio- Medical Electronics Co. Ltd., Shenzhen, China) and analyzed using FlowJo Version 10.1 software. For the analysis of tumor-associated macrophages, cell suspensions of lymph nodes were obtained by grinding and filtering tissues through 0.4-μm cell strainers (BD Biosciences, USA) in PBS. Cells were then transferred to a fresh tube for centrifugation at 1000× g for 5 min. The cell pellet was incubated in red blood cell lysis buffer (Lonza) for 1 min at room temperature, diluted in PBS, and centrifuged for 1000× g for 5 min. The cell pellet was incubated with fluorescent-conjugated antibodies (dilution 1:50 in PBS) for 15 min at 4 °C in the dark, washed, and analyzed by flow cytometry. The following antibodies were used: F4/80 (6F12) from Miltenyi; CD11b (M1/70.15.5) and Gr1 (RB6–8C5) from BD Pharmingen.

Vecchio E., Fiume G., Mignogna C., Iaccino E., Mimmi S., Maisano D., Trapasso F, & Quinto I. (2020). IBTK Haploinsufficiency Affects the Tumor Microenvironment of Myc-Driven Lymphoma in E-myc Mice. International Journal of Molecular Sciences, 21(3), 885.

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Analyzing Cell Cycle Dynamics via Flow Cytometry

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All flow cytometry was performed on a FACSCanto cytometer (BD Biosciences). Mouse cell surface marker staining was determined with the Lineage Cocktail kit and streptavidin-APC-Cy7 in combination with CD44-PE, CD49f-FITC, and CD24-APC antibodies (BD Biosciences). For co-culturing experiments, 2×10⁵ cells of each genotype were plated in 60 mm dishes, grown overnight, then treated with 100 μM BrdU for 35 minutes before staining with the APC BrdU kit (BD Biosciences). BrdU incorporation was measured in GFP− and GFP+ cells.

Privette Vinnedge L.M., Benight N.M., Wagh P.K., Pease N.A., Nashu M.A., Serrano-Lopez J., Adams A.K., Cancelas J.A., Waltz S.E, & Wells S.I. (2014). The DEK oncogene promotes cellular proliferation through paracrine Wnt signaling in Ron receptor positive breast cancers. Oncogene, 34(18), 2325-2336.

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Multiparameter Flow Cytometry of Hematopoietic Cells

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All antibody staining was performed in HBSS buffer (Corning #21021CV) containing Pen/Strep (100 Units/mL; Fisher Scientific #MT30002CI), HEPES (10uM; Life Technologies #15630080) and FBS (2%; Sigma #14009C). Briefly, bone marrow (BM) cells isolated from tibias, femurs, and iliac crests were combined for calculating total BM from each mouse. 1.0×10⁸ cells/mL were suspended in complete HBSS and incubated on ice for 20 min with the desired antibodies listed in the table below. The following antibodies were used at 1:100 dilutions - anti-mouse CD45-BV605 (clone 30-F11; BioLegend #103139), anti-human CD45-APC (clone 2D1; BioLegend #368512), anti-human CD71-APCcy7 (clone CY1G4; BioLegend #334110), anti-human CD235a-PE (clone HI264; BioLegend #349105), anti-human CD3-FITC (clone OKT3; BioLegend #317306), anti-human CD19-PECy7 (clone HIB19; BioLegend #302215), anti-human CD33-BV421 (clone WM53; BD #565949), anti-human CD34-PE (clone 561; BioLegend #343606), anti-human CD90-PECy7 (clone 5E10; BioLegend #328124), anti-human CD45RA-BV421 (clone HI100; BioLegend #304130), anti-human CD38-FITC (clone HB7; Invitrogen #11-0388-42), anti-human Lineage cocktail-APC (BioLegend #348803), anti-human CD45-biotin (clone HI30; BioLegend #304004), and Streptavidin-APCcy7 (BD #554063).

Celik H., Krug E., Zhang C.R., Han W., Issa N., Koh W.K., Bjeije H., Kukhar O., Allen M., Li T., Fisher D.A., Fowles J.S., Wong T.N., Stubbs M.C., Koblish H.K., Oh S.T, & Challen G.A. (2021). A Humanized Animal Model Predicts Clonal Evolution and Therapeutic Vulnerabilities in Myeloproliferative Neoplasms. Cancer discovery, 11(12), 3126-3141.

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