Apc conjugated streptavidin

Manufactured by BioLegend

Sourced in United States

APC-conjugated streptavidin is a fluorescent labeling reagent. It is composed of the protein streptavidin conjugated to the fluorescent dye allophycocyanin (APC). Streptavidin has a high affinity for the small molecule biotin, allowing it to be used to detect and localize biotinylated molecules.

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Lab products found in correlation

Facsverse, by BD (4 mentions) Biotinylated protein l, by ACROBiosystems (2 mentions) Pe labeled anti cd45ra, by BioLegend (2 mentions) Pe cy7 labeled anti cd62l, by BioLegend (2 mentions) Ez link sulfo nhs biotin, by Thermo Fisher Scientific (2 mentions) Facsaria 2, by BD (2 mentions) Biotinylated rabbit control igg, by Southern Biotech (2 mentions) Control rabbit igg, by Abcam (2 mentions) Lightning link rapid type a biotin conjugation kit, by Abcam (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Facsdiva software, by BD (1 mentions) Cd3ε clone 145 2c11, by BioLegend (1 mentions) Cd8α clone 53 6.7, by Cytek Biosciences (1 mentions) Ter119 clone ter 119, by BioLegend (1 mentions) Cd5 clone 53 7.3, by BioLegend (1 mentions) F4 80 clone bm8.1, by Cytek Biosciences (1 mentions) Percp cy5.5 conjugated cd25 clone pc61, by BioLegend (1 mentions) Apc cy7 conjugated cd45, by BioLegend (1 mentions) Nk1.1 clone pk136, by BioLegend (1 mentions)

25 protocols using apc conjugated streptavidin

Tracking Adoptive T Cell Trafficking

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Mice with MAV that had received pmel adoptive transfer were placed under a heat lamp for 5 minutes, and then injected with 3 ug of biotin-conjugated anti-CD45 anti-mouse antibody (30-F11, Biolegend), followed three minutes later by euthanasia and whole-body PBS perfusion through the cardiac ventricle. APC-conjugated streptavidin (Biolegend) was used to detect anti-CD45 labeled Thy1.1⁺ pmel cells, as evidence of their presence in the vascular space, in conjunction with Ab staining as described above.

Molodtsov A.K., Khatwani N., Vella J.L., Lewis K.A., Zhao Y., Han J., Sullivan D.E., Searles T., Preiss N.K., Shabaneh T.B., Zhang P., Hawkes A., Malik B.T., Kolling F I.V., Usherwood E.J., Wong S.L., Phillips J.D., Shirai K., Angeles C.V., Yan S., Curiel T.J., Huang Y.H., Cheng C, & Turk M.J. (2021). Resident memory CD8+ T cells in regional lymph nodes mediate immunity to metastatic melanoma. Immunity, 54(9), 2117-2132.e7.

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Flow Cytometric Analysis of Recombinant Antigen Binding

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Cells were collected and washed once with FACS buffer (PBS/5% FBS /0.02% sodium azide). Tagged recombinant target antigens (bio-hTEM1 (Fierle et al. 2019), bio-hCD19 [AAs 20–291; Acro Biosystems, cat. CD9-H82E9], bio-hMSLN [AAs 296-580; Acro Biosystems, cat. MSN-H82E9], hMSLN-his [AAs 1-286; SinoBiologics, cat. 13,128-H08H-50], hMSLN [AAs 296-580; R&D Systems, cat. 3265-MS-050]) were added to the cells at 1 µg/ml in FACS buffer. After 30 min of incubation on ice followed by three wash steps with 100 µl FACS buffer, antigen binding was revealed using either APC-conjugated streptavidin (1:2000; Biolegend cat. 405,207) or anti-Histag-Alexa647 (1/1000; Genscript, cat. A01802-100), diluted in FACS buffer. Stained cells were incubated for 30 min on ice and washed a further three times. Immediately before data acquisition, dead cells were stained with 4′,6-Diamidino-2-phenylindole (DAPI, 1:2000 dilution). Data was acquired using an LSR-II flow cytometer equipped with FACSDIVA software (BD Biosciences). Data analysis and plotting were carried out using FlowJo v10 (FlowJo LLC).

Fierle J.K., Abram-Saliba J., Atsaves V., Brioschi M., de Tiani M., Reichenbach P., Irving M., Coukos G, & Dunn S.M. (2022). A cell-based phenotypic library selection and screening approach for the de novo discovery of novel functional chimeric antigen receptors. Scientific Reports, 12, 1136.

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Identification of Lung ILC2 Cells

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Lung ILC2 cell identification was performed as described previously (47 (link)). Lung tissues were digested in 8 mL RPMI 1640 containing liberase (50 μg/mL) and DNase I (1 μg/mL) for approximately 40 minutes at 37°C. Cell suspensions were filtered through 70 μm cell strainers and washed once with RPMI 1640. For ILC2 cell identification, total lung cell suspensions were blocked with 2.4G2 antibodies and stained with lineage cocktail mAbs: CD3ε (clone 145-2C11) (BioLegend, 100304), CD4 (clone GK1.5) (BioLegend, 100404), CD8α (clone 53-6.7) (Tonbo Biosciences, 30-0081-U500), CD11c (clone N418) (BioLegend, 117304), FceRIα (clone MAR-1) (BioLegend, 134304), NK1.1 (clone PK136) (BioLegend, 108704), CD19 (clone 6D5) (BioLegend, 115504), TER119 (clone TER-119) (BioLegend, 116204), CD5 (clone 53-7.3) (BioLegend, 100604), F4/80 (clone BM8.1) (Tonbo Biosciences, 30-4801-U500), Ly6G (clone RB6-8C5) (Tonbo Biosciences, 30-5931-U500), APC-conjugated streptavidin (BioLegend, 405207), PE-conjugated T1/ST2 (clone DIH9) (BioLegend, 145304), PerCP-Cy5.5-conjugated CD25 (clone PC61) (BioLegend, 102030), V450-conjugated Sca-1 (clone D7) (BD Biosciences, 560653), PE-Cy7-conjugated KLRG1 (clone 2F1/KLRG1) (BioLegend, 138416), APC-Cy7-conjugated CD45, and Fixable Viability Dye eFluor 506.

She L., Barrera G.D., Yan L., Alanazi H.H., Brooks E.G., Dube P.H., Sun Y., Zan H., Chupp D.P., Zhang N., Zhang X., Liu Y, & Li X.D. (2021). STING activation in alveolar macrophages and group 2 innate lymphoid cells suppresses IL-33–driven type 2 immunopathology. JCI Insight, 6(3), e143509.

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Murine Splenocyte Immune Profiling

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Mouse splenocytes were cultured in a complete RPMI-1640 medium (GenDEPOT, Katy, TX, USA) supplemented with fetal bovine serum (10%, Hyclone, South Logan, UT, USA) and 1% penicillin/streptomycin (Welgene, Gyeongsan, Korea). Anti-mouse TCR-β-PE, CD45.1-Percp Cy5.5, CD3-APC Cy7, CD8-FITC, CD44-PE, and IFN-γ-FITC antibodies as well as CFSE cell proliferation tracing dye were purchased from Tonbo Bioscience (San Diego, CA, USA). Anti-mouse TNF-α-PE Cy7, APC-conjugated streptavidin, and PE-conjugated streptavidin were purchased from Biolegend (San Diego, CA, USA). Gp^33-41 class I pMHC tetramer was provided by the NIH Tetramer Core Facility (Atlanta, GA, USA).

Lim Y., Kim S., Kim S., Kim D.I., Kang K.W., Hong S.H., Lee S.M., Koh H.R, & Seo Y.J. (2020). n-3 Polyunsaturated Fatty Acids Impede the TCR Mobility and the TCR–pMHC Interaction of Anti-Viral CD8+ T Cells. Viruses, 12(6), 639.

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Analyzing Sindbis Virus Envelope Protein Expression

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293T cells were transfected with expression vectors of wild-type Sindbis virus envelope protein, 2.2 1L1L, E2 71 AV, E2 71 STAV, E2 71 eMA, or E2 71 mSAH, using TransIT LT1. Two days post-transfection, expression of the envelope proteins was analyzed by staining the transfected cells with rabbit anti-Sindbis virus antibody, followed by staining with Alexa 488-conjugated goat anti-rabbit IgG antibody (ThermoFisher Scientific). The binding of biotin was analyzed by staining the transfected cells with biotinylated FITC. Expression of hTfR1 and mCD34 on Jurkat and NIH3T3 cells was analyzed by staining cells with either anti-hTfR1, mCD34, or isotype control antibodies, followed by staining with Alexa 488-conjugated streptavidin (ThermoFisher Scientific). Expression of hTfR1 and EGFR on HeLa and Jurkat cells was analyzed by staining the cells with APC-conjugated anti-hTfR1, EGFR, or its isotype control antibody. Binding of EGFR to HeLa and Jurkat cells was analyzed by staining the cells with biotinylated EGF, followed by staining with APC-conjugated streptavidin (Biolegend). Flow cytometric data were acquired by FACScan (BD) upgraded with a red laser (Cytek, Fremont, CA) and analyzed by FCSExpress 5 (De Novo Software, Los Angeles, CA).

Situ K., Chua B.A., Bae S.Y., Meyer A, & Morizono K. (2018). Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules. Virology, 525, 170-181.

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Quantifying KIT Internalization Kinetics

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Internalization assay for KIT was performed as described previously [26] (link). Briefly, WT and CALM^−/− MEFs both engineered to express KIT were cultured with biotinylated SCF (R&D systems, Minneapolis, MN) for 60 min, and further incubated with the APC-conjugated streptavidin (Biolegend, San Diego, CA) for 30 min at 4°C. Then, these cells were incubated at 37°C up to 20 min to allow internalization. After stripping unincorporated SCF with acidic buffer (20 mM MES pH 5, 130 mM NaCl, 2 mM CaCl2 and 0.1% BSA), relative amount of internalized SCF-KIT complex was evaluated from the fluorescence intensity by FACS at the indicated times compared with the initial amount of membrane KIT.

Rai S., Tanaka H., Suzuki M., Ogoh H., Taniguchi Y., Morita Y., Shimada T., Tanimura A., Matsui K., Yokota T., Oritani K., Tanabe K., Watanabe T., Kanakura Y, & Matsumura I. (2014). Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells. PLoS ONE, 9(10), e109441.

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Murine MHC Class I Tetramer Production

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Expression of recombinant murine MHC class I H-2L^d heavy chain and of human β2-microglobulin was performed according to established protocols. Refolding of the MHC class I complex with the AH1 peptide was followed by biotinylation with MBP-BirA and size exclusion chromatography (Superdex S75 10/300 GL, GE Healthcare). Assembled monomers were stored in 16% glycerol at -20 ºC. MHC class I tetramers were produced by addition of APC-conjugated streptavidin (Biolegend) to the monomers at a final molecular ratio of 4:1. Plasmids of the H-2L^d heavy chain and of human β2-microglobulin were a kind gift of Prof. A. Oxenius (ETH Zürich, Switzerland). The AH1 (SPSYVYHQF) peptide was ordered from Biomatik.

Probst P., Stringhini M., Ritz D., Fugmann T, & Neri D. (2018). Antibody-based delivery of TNF to the tumor neo-vasculature potentiates the therapeutic activity of a peptide anti-cancer vaccine. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(2), 698-709.

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Characterization of CAR T Cell Phenotypes

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5 × 10⁵human T cells were infected by the lentivirus mentioned above. To draw cell growth curves, live T cells were counted using trypan blue staining and Automated Cell Counter (Thermo Fisher, Countess™ 3). Expression of CD19-, BCMA-, BC19- or 19BC CAR was evaluated by Biotinylated protein L (Acro Biosystems, Cat: RPL-P814R, Lot: BL11R-76EF1-GY,1:400 dilution) and APC--conjugated Streptavidin (Biolegend, Cat: 405207, Lot: B388667, 1:100 dilution). PE-labeled anti-CD45RA (Biolegend, Clone: HI100, Cat: 304107, Lot: B378521, 1:100 dilution) and PE-Cy7-labeled anti-CD62L (Biolegend, Clone: DREG-56, Cat: 304821, Lot: B373156, 1:100 dilution) were employed to determine phenotypes of infusion products of CAR T, including naïve (CD45RA⁺ CD62L⁺), central memory (CD45RA⁻ CD62L⁺), effector memory (CD45RA⁻ CD62L⁻), and effector (CD45RA⁺ CD62L⁻) T cells.

Shi M., Wang J., Huang H., Liu D., Cheng H., Wang X., Chen W., Yan Z., Sang W., Qi K., Li D., Zhu F., Li Z., Qiao J., Wu Q., Zeng L., Fei X., Gu W., Miao Y., Xu K., Zheng J, & Cao J. (2024). Bispecific CAR T cell therapy targeting BCMA and CD19 in relapsed/refractory multiple myeloma: a phase I/II trial. Nature Communications, 15, 3371.

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Biotinylation and Stv-UCNP Coupling of CAR T-cells

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Jurkat or human CD8⁺ T-cells expressing WT CAR or LiCAR, as well as Jurkat T cells as control, were washed three times in cold PBS to deplete residual free amine-containing buffers. Cells were then suspended at a concentration of 2.5 x 10⁷ cells/ml. Subsequently, 200 ul of 10 mM EZ-Link Sulfo-NHS-Biotin (#A39256, Thermo Fisher Scientific) was added to 1 ml of cell suspension. The mixture was incubated at 4°C for 30 minutes. The cells were washed three times with 100 mM glycine in PBS. Biotinylated cells were further incubated with APC-conjugated streptavidin (#405207, BioLegend) to evaluate the biotinylation efficiency by flow cytometry. To prepare for Stv-UCNP-CAR T-cell mixture, 0.1 ml Stv-UCNPs (1.5 mg/ml) was mixed with 1 x 10⁷ biotinylated hCD8⁺ T-cells expressing WT CAR or LiCAR, and then incubated at 4°C for 30 min. Unbound St-UCNPs were washed away by centrifuging the mixture at 700 x g for 5 minutes. UCNP-coupled CAR T-cells were re-suspended in PBS and administered into mice via tail vein injection.

Nguyen N.T., Huang K., Zeng H., Jing J., Wang R., Fang S., Chen J., Liu X., Huang Z., You M.J., Rao A., Huang Y., Han G, & Zhou Y. (2021). Nano-Optogenetic Engineering of CAR T-cells for Precision Immunotherapy with Enhanced Safety. Nature nanotechnology, 16(12), 1424-1434.

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Comprehensive Phenotyping of T Cell Subsets

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Peptide-HLA monomers were obtained from ImmunAware (Copenhagen, Denmark) with the exceptions of A*11:01-ACQGVGGPSHK (Dr. Masafumi Takiguchi, Kumamoto University, Japan) and A*26:01-EVIPMFSAL (MBL International). Tetramers were produced by multimerization with APC-conjugated streptavidin (Biolegend) as per manufacturer’s protocol and stored at 4°C for a maximum of 4 weeks prior to use. Staining was performed using individual pH LA tetramers at 4°C prior to surface marker staining to prevent cell activation and steric interference. Where indicated, cells were also stained with BUV395-conjugated anti-CD8 (clone RPA-T8, BD Biosciences), APC/Cy7-conjugated anti-CD45RA (clone HI100, Biolegend) and FITC-conjugated anti-CD62L (clone DREG-56, Biolegend) and Live/Dead Violet viability dye (Thermo Fisher); or with BUV395-conjugated anti-CD8 (clone RPA-T8, BD Biosciences), BV605-conjugated anti-PD1 (clone EH12.2H7, Biolegend), AlexaFluor 488-conjugated anti-TIM3 (clone 344823, R&D Systems), PE/Dazzle 594-conjugated anti-TIGIT (clone A15153G, Biolegend), PE/Cy7-conjugated anti-CD160 (clone BY55, Biolegend), PerCP/Cy5.5-conjugated anti-2B4 (clone C1.7, Biolegend), BV650-conjugated anti-CD39 (clone TU66, BD Biosciences), BV510-conjugated anti-CD57 (clone HNK-1, Biolegend) and APC/Cy7-conjugated anti-CD28 (clone CD28.2, Biolegend) and analyzed by flow cytometry.

Collins D.R., Urbach J.M., Racenet Z.J., Arshad U., Power K.A., Newman R.M., Mylvaganam G.H., Ly N.L., Lian X., Rull A., Rassadkina Y., Yanez A.G., Peluso M.J., Deeks S.G., Vidal F., Lichterfeld M., Yu X.G., Gaiha G.D., Allen T.M, & Walker B.D. (2021). Functional impairment of HIV-specific CD8+ T cells precedes aborted spontaneous control of viremia. Immunity, 54(10), 2372-2384.e7.

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