Streptavidin alexa 647

Manufactured by Thermo Fisher Scientific

Sourced in United States

Streptavidin Alexa 647 is a fluorescent conjugate of streptavidin and the Alexa Fluor 647 dye. Streptavidin is a tetrameric protein that binds to the small molecule biotin with high affinity. The Alexa Fluor 647 dye provides a strong red fluorescent signal that can be detected using standard fluorescence detection methods.

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Alexa 647 (424 citations) Streptavidin Alexa Fluor 647 (66 citations) Alexa Fluor 647-conjugated streptavidin (38 citations) Alexa 647-conjugated streptavidin (17 citations) Streptavidin 647 (7 citations) Streptavidin Alexa Fluor® 647 Conjugate (5 citations) Alexa-488 or Alexa 647-streptavidin (2 citations) Alexa Fluor-647-labelled streptavidin (2 citations)

28 protocols using streptavidin alexa 647

Measuring MR1 Expression in BEAS-2B Cells

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BEAS-2B:MR1_KO cells, either untransfected, or transfected with pCI empty vector, pCI_MR1AGFP, and/or pCI_MR1BRFP, were plated into 6 well tissue culture plates overnight. The next day, the cells were treated with 100 µM 6-FP versus an equivalent volume of 0.01 M NaOH. After 16 h, the cells were harvested on ice and split into two groups for primary staining and isotype control staining. Primary staining was done with an antibody against MR1 (Clone 26.5, gift from Ted Hansen, biotinylated by Biolegend, San Diego, USA) at 1:100 for 40 min on ice in the presence of 2% human serum, 2% goat serum, and 0.5% FBS. Biotinylated mouse anti IgG2A (Biolegend, San Diego, USA) served as the isotype control. After washing, streptavidin-Alexa 647 (ThermoFisher Scientific, Waltham, USA) was added for 40 min on ice. Cells were washed, fixed in 1% PFA and analyzed by flow cytometry on a BD Fortessa, or BD Symphony and data were analyzed on FlowJo (TreeStar, Ashland, USA).

Narayanan G.A., Nellore A., Tran J., Worley A.H., Meermeier E.W., Karamooz E., Huber M.E., Kurapova R., Tafesse F.G., Harriff M.J, & Lewinsohn D.M. (2020). Alternative splicing of MR1 regulates antigen presentation to MAIT cells. Scientific Reports, 10, 15429.

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Magnetic Cell Isolation via Insulin Staining

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As previously described [12 (link), 26 (link)], cells were stained at 4°C with 0.1 μg insulin– biotin per 10⁶ cells per 100 μl and antibodies (as described below) for 30 min, fixed with 2% (wt/vol.) formaldehyde and incubated with streptavidin–Alexa⁶⁴⁷ (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min. Cells were then washed and incubated with anti-Cy5/anti-Alexa⁶⁴⁷ microbeads (Miltenyi) for 10 min and passed over magnetised LS columns (Miltenyi). Columns were washed three times, and bound cells were eluted in 6 ml of MACS buffer.

Smith M.J., Hinman R.M., Getahun A., Kim S., Packard T.A, & Cambier J.C. (2018). Silencing of high-affinity insulin-reactive B lymphocytes by anergy and impact of the NOD genetic background in mice. Diabetologia, 61(12), 2621-2632.

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Mannose-Cluster Internalization in moDCs

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Immature day 5 moDCs were harvested and washed with cold HBSS (Thermo Fischer), after which half of the moDCs were gently fixed for 20 min at RT with 1% PFA in PBS. Afterwards, 20 μM of the different biotinylated mannose-clusters in cold HBSS were added. The moDCs were incubated for 1 h on ice, and washed in cold HBSS. Subsequently, warm HBSS was added to the cells, and cells were incubated at 37°C in a shaking heating block. At the indicated time points, a sample of the cells was taken and put on ice. After the last time point, the cells were stained with Streptavidin-Alexa647 (Thermo Fisher), measured using flow cytometry (CyAn™ ADP with Summit™ Software), and further analyzed using FlowJo v10. The same procedure was used for internalization with pHrodo^TM Red Avidin (Thermo Fischer). The different biotinylated mannose-clusters were however incubated with pHrodo-Avidin (ratio 2:1) for 15 min at 37°C, prior to moDC exposure. The fluorescence upon internalization was measured using flow cytometry (BD LSRFortessa™ X-20 with FACSDiva Software), and further analyzed using FlowJo v10.

Li R.J., Hogervorst T.P., Achilli S., Bruijns S.C., Arnoldus T., Vivès C., Wong C.C., Thépaut M., Meeuwenoord N.J., van den Elst H., Overkleeft H.S., van der Marel G.A., Filippov D.V., van Vliet S.J., Fieschi F., Codée J.D, & van Kooyk Y. (2019). Systematic Dual Targeting of Dendritic Cell C-Type Lectin Receptor DC-SIGN and TLR7 Using a Trifunctional Mannosylated Antigen. Frontiers in Chemistry, 7, 650.

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Quantifying Mannoside Binding on Langerhans Cells

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The cells were harvested and washed with cold (4°C) HBSS, before preincubation with 20 μg/mL anti-CD207 [clone 10E2, in-house (de Witte et al., 2007 (link))] for 45 min on ice in the blocking condition, with 1% PFA in PBS for 20 min on room temperature (RT) in the gently fixated condition, or without pretreatment; 20 μM of the hexavalent biotinylated mannosides was added to the cells in cold HBSS and incubated for 1 h at 4°C. The unbound clusters were washed away with cold HBSS. Warm (37°C) HBSS was added to the cells followed by incubation at 37°C in a shaking heating block. At the indicated time points, a sample was taken and put on ice. The cells were then stained with streptavidin-Alexa647 (Thermo Fisher), and the fluorescence was quantified with flow cytometric analysis (CyAn^TM APD with Summit^TM software) and analyzed with FlowJo v10.

Li R.J., Hogervorst T.P., Achilli S., Bruijns S.C., Spiekstra S., Vivès C., Thépaut M., Filippov D.V., van der Marel G.A., van Vliet S.J., Fieschi F., Codée J.D, & van Kooyk Y. (2020). Targeting of the C-Type Lectin Receptor Langerin Using Bifunctional Mannosylated Antigens. Frontiers in Cell and Developmental Biology, 8, 556.

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Comparative Profiling of Notch1+ and Glast+ Cell Populations

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Three independent biological replicates were obtained for comparative profiling of purified P0 Notch1+, P3 Notch1+, P7 Notch1+, P14 Notch1+, and P14 Glast+ cells. Two independent biological replicates were obtained for comparative profiling of purified P7 Glast+, P21 Glast+ and P28 Glast+ cells. RNA was purified from samples using the Absolutely RNA® Nanoprep kit (Agilent Technologies, Santa Clara, CA). RNA samples were sent to Ocean Ridge Biosciences (Palm Beach Gardens, FL, USA) for processing using MEEBO microarrays. Biotin-labeled complementary RNA (cRNA) was prepared from the total RNA according to Van Gelder’s protocol [21 (link)]. Biotinylated cRNA samples were fragmented, diluted in a hybridization buffer, and loaded on to the MEEBO microarray slides (for more information on the MEEBO oligonucleotide set please refer to http://alizadehlab.stanford.edu/). The slides were hybridized for 16–18 hours in a Model 400 hybridization oven (Scigene, Sunnyvale, CA). After hybridization, the microarray slides were washed, stained with Streptavidin-Alexa-647 (ThermoFisher Scientific, Grand Island, NY), and scanned using an Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA).

Dvoriantchikova G., Seemungal R.J, & Ivanov D. (2019). Development and epigenetic plasticity of murine Müller glia. Biochimica et biophysica acta. Molecular cell research, 1866(10), 1584-1594.

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Ca2+ Flux Assay for Immune Cell Activation

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The Ca²⁺ flux assay was performed as previously described [25 (link)]. In brief, cell samples were suspended at a density of 10⁷ cells per ml in phosphate-buffered saline (PBS) and loaded with 2 mM Indo-1 AM (acetoxymethyl) for 30 min at 37 °C, followed by washing twice with culture RPMI. Cells were pre-warmed to 37 °C for 10 min before analysis and were kept at 37 °C in culture RPMI during the event collection. For cell stimulation, an HLA-A*02:01-L2 monomer was pre-refolded, biotinylated [26 (link)], and crosslinked with streptavidin Alexa 647 (Thermo Fisher Scientific) to form the antigen tetramer. Cells were then stimulated with monomers or tetramers. The mean fluorescence ratio of Indo-1 high to Indo-1 low was calculated using FlowJo (Version 10) kinetics program.

Wu L., Brzostek J., Sankaran S., Wei Q., Yap J., Tan T.Y., Lai J., MacAry P.A, & Gascoigne N.R. (2021). Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model. Cancers, 13(4), 867.

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Calcium Flux Assay for Cell Stimulation

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The calcium flux assay was performed as previously described.⁵⁷ (link) In brief, cells were suspended at 10⁷/mL in PBS (PBS) and loaded with 2 μM Indo-1 AM for 30 min at 37°C, followed by washing twice with cRPMI. Cells were pre-warmed to 37°C for 10min before analysis and kept at 37°C during analysis. For cell stimulation, an HLA-A2-L2 monomer was pre-refolded, biotinylated⁵⁸ (link) and crosslinked with streptavidin Alexa 647 (ThermoFisher) to form tetramer, that was used to stimulate cells. Mean fluorescence ratio of Indo-1^high/Indo-1^low was calculated using FlowJo kinetics program.

Wu L., Brzostek J., Sakthi Vale P.D., Wei Q., Koh C.K., Ong J.X., Wu L.Z., Tan J.C., Chua Y.L., Yap J., Song Y., Tan V.J., Tan T.Y., Lai J., MacAry P.A, & Gascoigne N.R. (2023). CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance. Cell Reports Medicine, 4(2), 100917.

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Immunofluorescent Analysis of Murine Thymus

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Adult thymus tissues from host mice were frozen, 7-μm sections were cut, and then fixed in acetone and stained with the following Abs: the mTEC marker ERTR5 (a gift from Dr. W. van Ewijk, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan), detected with Alexa Fluor 594 goat anti-rat IgM (Invitrogen), biotinylated anti-CD45.1 (clone A20; eBioscience), detected by Streptavidin Alexa 647 (Invitrogen), FITC anti-CD45.2 (clone 104; eBioscience), followed by rabbit anti-FITC (Invitrogen), and donkey anti-rabbit 488 (Invitrogen), biotinylated anti-CD8 (clone YTS156.7.7; BioLegend), detected by Streptavidin Alexa 488 (Invitrogen), and purified anti-CD4 (clone GK 1.5; eBioscience), conjugated to Alexa 647 (Invitrogen). Images were obtained using a LSM 780 microscope and analyzed using LSM software. (Zeiss). For confocal quantitation, a minimum of three separate thymus sections at least 10 sections apart were analyzed for each mouse. Calculation of cell frequency within a defined area was performed as previously described (34 (link)), where square fields of 100 × 100μm were arbitrarily set within either medullary or cortical regions, at least 100 μm from the CMJ.

Cowan J.E., McCarthy N.I., Parnell S.M., White A.J., Bacon A., Serge A., Irla M., Lane P.J., Jenkinson E.J., Jenkinson W.E, & Anderson G. (2014). Differential Requirement for CCR4 and CCR7 during the Development of Innate and Adaptive αβT Cells in the Adult Thymus. The Journal of Immunology Author Choice, 193(3), 1204-1212.

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Dendritic Spine Imaging Using Biocytin

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As described in previous study, whole-cell recordings with voltage clamp at –70 mV for about 10–15 mins. The cesium methanesulfonate internal solution was made as described above with 2 mg/mL Biocytin (Sigma Cat#B4261). Then slices were transferred to 4%PFA/PBS and fixed one hour in room temperature. Slices were washed 3x5 min with PBS, permeabilized in 0.3% Triton-X100/PBS for 30 min, and blocked in 5% normal goat serum (NGS)/0.1% Triton-X100/PBS at room temperature for 1 hr. Subsequently, slices were incubated in Streptavidin Alexa 647 (Invitrogen Cat#S32357) diluted 1:1000 in 5% NGS/0.1% Triton-X100/PBS at 4 °C overnight, washed 5x5 min with PBS and mounted with 0 thickness coverglass (Assistent Cat#01105209). Images were acquired using a Nikon A1 Eclipse Ti confocal microscope with 20 x and 100 x objectives, operated by NIS-Elements AR acquisition software. For spine imaging, Z-stacks were collected at 0.2 μm with 0.06 μm/pixel resolution, and 6–10 dendrites were analyzed per cell.

Dai J., Liakath-Ali K., Golf S.R, & Südhof T.C. (2022). Distinct neurexin-cerebellin complexes control AMPA- and NMDA-receptor responses in a circuit-dependent manner. eLife, 11, e78649.

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CD6 and CD6ΔD3 Tetramer Assay

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Recombinant extracellular WT‐CD6 (GenBank AAA86419.1) and CD6∆d3 (GenBank ABH04237.1) were produced and biotinylated as described elsewhere;13 amplification of CD6∆d3 was from pEGFP‐N1/CD6∆D3.14Tetramers were assembled by mixing 3 μg of biotinylated CD6 or CD6∆d3 with 0·75 μg of Streptavidin‐Alexa647 (Invitrogen) and incubating for 1 hr at 4° with agitation. The assembled mixture was added to 2·5 × 10⁵ cells and allowed to interact for 45 min on ice. Cells were washed and analysed by flow cytometry.
In blocking experiments, CD6 mAbs were added to the assembled tetramers and incubated for 20 min on ice before cell staining.

Santos R.F., Oliveira L., Brown M.H, & Carmo A.M. (2019). Domain‐specific CD6 monoclonal antibodies identify CD6 isoforms generated by alternative‐splicing. Immunology, 157(4), 296-303.

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