Allophycocyanin apc conjugated streptavidin

Manufactured by BD

Sourced in United States

Allophycocyanin (APC)-conjugated streptavidin is a fluorescent-labeled protein complex used in various biotechnology applications. Streptavidin is a protein derived from the bacterium Streptomyces avidinii and has a high affinity for the small molecule biotin. APC is a natural fluorescent protein from blue-green algae that serves as the fluorescent label. This product combines the binding properties of streptavidin with the fluorescent characteristics of APC, allowing for the detection and visualization of biotinylated molecules.

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6 protocols using allophycocyanin apc conjugated streptavidin

Bovine CD8+ T Cell Immunophenotyping

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Multi-colour flow cytometry analysis was performed as described previously [19 (link)]. Briefly, cells were incubated with a cocktail of monoclonal antibodies against rabbit CD4 (IgG2a, KEN-4), CD8 (IgG1, 12C.7) and IgM (IgG1, NRBM) on ice for 10 min. Cells were washed and further incubated for 10 min on ice with isotype-specific phycoerythrin (PE)-conjugated rat anti-mouse IgG1 (A85-1, BD) and biotinylated rat anti-mouse IgG2a (R19-15, BD) antibodies. After a third wash, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit T cells (KEN-5), washed with allophycocyanin (APC)-conjugated streptavidin (BD), and suspended in 7-AAD. Antibodies were from AbD-Serotec. Bovine CD8⁺ T cells were identified as previously described [18 (link)], and dead cells detected with BD Horizon Fixable Viability Stain 450 (BD biosciences). Data were acquired using a Fortessa X20 flow cytometer (BD) and analyzed using Flowjo v10.0.7 (Treestar).

Myster F., Gong M.J., Javaux J., Suárez N.M., Wilkie G.S., Connelley T., Vanderplasschen A., Davison A.J, & Dewals B.G. (2020). Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever. PLoS Pathogens, 16(3), e1008405.

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Comprehensive Cytometric Analysis of T Cell Subsets

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The following antibodies and reagents were used. FITC-conjugated anti-mouse CD45R/B220 (RA3-6B2, 5 μg ml⁻¹), FITC-conjugated anti-mouse CD4 (RM4–5, 5 μg ml⁻¹), biotinylated anti-mouse CD4 (RM4–5, 2.5 μg ml⁻¹), PE-conjugated anti-mouse CD8a (53-6.7, 1 μg ml⁻¹), PE-conjugated anti-mouse CD44 (IM7, 1 μg ml⁻¹), FITC-conjugated anti-mouse CD62L (MEL-14, 5 μg ml⁻¹), FITC-conjugated anti-mouse Vα11 (RR8-1, 5 μg ml⁻¹), FITC-conjugated anti-mouse Vα2 (B20.1, 5 μg ml⁻¹), biotinylated anti-mouse Vβ3 (KJ25, 2.5 μg ml⁻¹), biotinylated anti-mouse Vβ5 (MR9-4, 2.5 μg ml⁻¹), and allophycocyanin (APC)-conjugated streptavidin (0.1 μg ml⁻¹) or PerCP-5.5cyanine-conjugated streptavidin (0.5 μg ml⁻¹) were from BD Biosciences. Biotinylated anti-mouse CD90.2/Thy1.2 (30-H12, 0.25 μg ml⁻¹) was purchased from eBioscience. Before staining with the antibodies, cells were incubated for 10 min on ice with anti-Fcγ III/II receptor (2.4G2, 0.5 μg ml⁻¹; BD Biosciences) antibody to block Fc receptors. Flow cytometoric analyses were done on FACS Calibur (BD Biosciences).

Yamamura K., Uruno T., Shiraishi A., Tanaka Y., Ushijima M., Nakahara T., Watanabe M., Kido-Nakahara M., Tsuge I., Furue M, & Fukui Y. (2017). The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction. Nature Communications, 8, 13946.

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Isolation and Analysis of Mouse Liver Cells

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A single‐cell suspension from the mouse liver was obtained by a two‐step collagenase perfusion method and used for preparing hepatocytes and nonparenchymal cells (NPCs) by centrifugal separation as described.23 Aliquots of cells were blocked with anti‐Fc receptor antibody, costained with fluorescence‐conjugated and/or biotin‐conjugated antibodies (listed in Supporting Table S1) and then incubated with allophycocyanin (APC)‐conjugated streptavidin (BD Biosciences) if necessary. We detected 5‐ethynyl‐2′‐deoxyuridine (EdU) by using the Click‐iT Plus EdU Alexa Fluor 488 Cytometry Assay Kit (Life Technologies), following the manufacturer's instructions. The samples were analyzed by FACSCanto II (BD Biosciences) or sorted by Moflo XDP (Beckman‐Coulter). Dead cells were excluded by propidium iodide staining. HSCs were identified based on the vitamin A autofluorescence signal detected by a violet laser at 405 nm. Data were analyzed using FlowJo software.

Katsumata L.W., Miyajima A, & Itoh T. (2017). Portal fibroblasts marked by the surface antigen Thy1 contribute to fibrosis in mouse models of cholestatic liver injury. Hepatology Communications, 1(3), 198-214.

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Isolation and Purification of Murine Liver Cells

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Cells were isolated from murine livers as described previously (Okabe et al., 2009 (link)). Briefly, liver cells were dissociated by perfusion of collagenase solution. Non-parenchymal cells (NPCs) were prepared by removal of hepatocytes with repeated centrifugation at 100 g for 2 min. Then, NPCs were incubated with anti-FcR antibody for blocking non-specific binding, followed by with fluorescein isothiocyanate (FITC)-conjugated anti-EpCAM monoclonal antibody for 30 min on ice. After incubation with anti-FITC microbeads (1:10–100 dilution, Miltenyi Biotec, Bergisch Gladbach, Germany), EpCAM⁺ cells were enriched by autoMACS pro (Miltenyi Biotec). After MACS, cells were incubated with biotin-conjugated anti-Lutheran monoclonal antibody for 30 min on ice. After wash, cells were incubated with allophycocyanin (APC)-conjugated streptavidin (1:100–500 dilution, BD bioscience, NJ, USA) for 20 min on ice and analyzed or purified by fluorescence-activated cell sorting (FACS) using Moflo XDP (Beckman-Coulter, CA, USA) and BD FACSCanto II (BD bioscience). Dead cells were excluded by propidium iodide (Sigma-Aldrich, MO, USA) staining.

Miura Y., Matsui S., Miyata N., Harada K., Kikkawa Y., Ohmuraya M., Araki K., Tsurusaki S., Okochi H., Goda N., Miyajima A, & Tanaka M. (2018). Differential expression of Lutheran/BCAM regulates biliary tissue remodeling in ductular reaction during liver regeneration. eLife, 7, e36572.

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CAR and EGFR Expression Detection

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For CAR detection, cells were stained with biotinylated protein L (GenScript, Piscataway, NJ, USA), goat anti-mouse IgG, and anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by streptavidin-conjugated allophycocyanin (APC) (BD). The surface expression of EGFR and its mutants was detected by CFP and APC-conjugated cetuximab antibody (Novus Biologicals, Centennial, CO, USA). EGFRvIII expression was detected by anti-EGFRvIII antibody, clone DH8.3 (Santa Cruz Biotechnology, Dallas, TX, USA). Flow analysis done by LSRFortessa (BD) and data were analyzed by FlowJo software (BD).

Thokala R., Binder Z.A., Yin Y., Zhang L., Zhang J.V., Zhang D.Y., Milone M.C., Ming G.L., Song H, & O’Rourke D.M. (2021). High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms. Frontiers in Oncology, 11, 664236.

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Immunophenotyping of Microglia and Myeloid Cells

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Rabbit polyclonal antibodies (pAbs) to Iba1 were obtained from Wako (Osaka, Japan). Biotin-conjugated Armenian hamster monoclonal antibody (mAb) to mouse CD11c (clone HL3), PerCP-Cy5.5 conjugated rat mAb to CD45 (30-F11), FITC-conjugated rat mAb to mouse CD11b (clone M1/70), rat mAb to mouse CD16/CD32 (clone 2.4G2), and streptavidin-conjugated allophycocyanin (APC) were from BD Pharmingen (San Diego, CA). PE conjugated rat mAb to mouse CD172a (SIRPα) (clone P84) was from eBioscience (San Diego, CA). PE conjugated and unconjugated rat mAbs to CD68 (clone FA-11) and PE conjugated rat mAb to mouse CD14 (clone Sa14-2) were from BioLegend (San Diego, CA). PE-conjugated recombinant antibody (Ab) to Dectin-1 (REA154) and PE-conjugated recombinant Ab to CD47 (REA170) were from Miltenyi Biotec (Bergisch Gladbach, Germany). 4',6-Diamidino-2-phenylindole (DAPI) was obtained from Nacalai Tesque (Kyoto, Japan). Rat mAb to myelin basic protein (MBP) (clone 12) was from Merck Millipore (Billerica, MA). Rabbit pAb to Olig2 (18953) was from Immuno-Biological Laboratories (Gunma, Japan).

Sato-Hashimoto M., Nozu T., Toriba R., Horikoshi A., Akaike M., Kawamoto K., Hirose A., Hayashi Y., Nagai H., Shimizu W., Saiki A., Ishikawa T., Elhanbly R., Kotani T., Murata Y., Saito Y., Naruse M., Shibasaki K., Oldenborg P.A., Jung S., Matozaki T., Fukazawa Y, & Ohnishi H. (2019). Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter. eLife, 8, e42025.

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