Streptavidin alexa fluor 647

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Streptavidin Alexa Fluor 647 is a fluorescently-labeled protein that binds with high affinity to the small molecule biotin. It can be used in various biological applications as a detection reagent.

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Alexa Fluor 647 (1520 citations) Alexa 647 (424 citations) Alexa Fluor 647-conjugated streptavidin (38 citations) Streptavidin Alexa 647 (28 citations) Alexa Fluors (21 citations) Streptavidin 647 (7 citations) Streptavidin Alexa Fluor® 647 Conjugate (5 citations) Alexa Fluor-647-labelled streptavidin (2 citations)

66 protocols using streptavidin alexa fluor 647

Isolation of ST6GAL1-overexpressing CHO Clones

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The enriched Zeocin resistant stable pool derived from CHOZN^® GS host cell line that overexpressed ST6GAL1 was single-cell cloned using a FACSAria™ III cell sorter. The cells with top 5% FITC-SNA fluorescence was plated at 1 cell/well in 96-well plates, with 200 μL per well of Ham’s F-12 supplemented with 6 mM L-glutamine and 10% fetal bovine serum (FBS). One plate was plated using unstained cells based on forward and side scatter and no fluorescence as control for clonal outgrowth.
Biotinylated Maackia amurensis lectin II (MALII, Vector Labs, Burlingame, CA) at a final concentration of 5 μg/mL for 15 min at 25°C was incubated with 5 × 10⁵ cells to stain for α2,3-linked sialic acid. Cells were washed twice with 1 × PBS supplemented with 0.1% bovine serum albumin (BSA) and incubated for 15 min at 25°C with 0.5 μg/mL streptavidin-Alexa Fluor647 (Life Technologies, Eugene, OR) and 10 μg/mL FITC-SNA. The cells were washed twice with 1 × PBS supplemented with 0.1% BSA before responded for two-color FACS analysis on a MACSQuant^® Analyzer (Miltenyi Biotec, San Diego, CA).

Lin N., Mascarenhas J., Sealover N.R., George H.J., Brooks J., Kayser K.J., Gau B., Yasa I., Azadi P, & Archer-Hartmann S. (2015). Chinese Hamster Ovary (CHO) Host Cell Engineering to Increase Sialylation of Recombinant Therapeutic Proteins by Modulating Sialyltransferase Expression. Biotechnology progress, 31(2), 334-346.

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Quantitative Immunofluorescence of T Cell Phenotypes

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Immunofluorescence of T cells was performed as previously described (10 (link)) with the following antibodies: anti-β-tubulin (T8328) (Sigma); anti-IL-2Rα (PC61.5), anti-T-bet (4B10), anti-LFA-1 (M17/4) (eBioscience); anti-proteasome 20S C2 (ab3325) (Abcam); anti-IFNγR (2E2) (Biolegend); and anti-mouse Alexa Fluor 488, anti-rat Alexa Fluor 488, anti-mouse Alexa Fluor 647, anti-rabbit Alex Fluor 647, streptavidin Alexa Fluor 647, and anti-rat Alexa Fluor 647 (Life Technologies). DAPI (Life Technologies) was used to detect DNA. Pre-mitotic cells were identified by a single microtubule organizing center (MTOC) with β-tubulin staining, mitotic blasts were identified by the presence of two MTOCs, and cells undergoing cytokinesis were identified by dual nuclei and pronounced cytoplasmic cleft by brightfield. Acquisition of image stacks was performed as previously described (10 (link)) using a FV1000 laser scanning confocal microscope (Olympus). The volume of 3D pixels (voxels) containing the designated protein fluorescence was quantified within each hemisphere or within nascent daughter in cytokinetic cells as previously described (10 (link)) using ImageJ software.

Metz P.J., Arsenio J., Kakaradov B., Kim S.H., Remedios K.A., Oakley K., Akimoto K., Ohno S., Yeo G.W, & Chang J.T. (2015). Regulation of Asymmetric Division and CD8+ T Lymphocyte Fate Specification by PKCζ and PKCλ/ι. Journal of immunology (Baltimore, Md. : 1950), 194(5), 2249-2259.

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Intracellular Antibody Quantification in CHO Cells

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CHO cells were fixed with ice-cold 70% (v/v) ethanol on day 1 and day 10 of semi-continuous perfusion experiment. For intracellular HC and LC product analysis, 10⁶ cells were stained with biotinylated polyclonal anti-human gamma- (Life technologies, no. A18821), anti-human kappa- (Antibodies-online, no. ABIN375958), or anti-human lambda-chain antibody (Novus Biologicals, no. NB100-62142) and conjugated with streptavidin-Alexa Fluor 647 (Life Technologies, no. A21244) according to the protocol published in Reinhart et al. (2014 (link)). Measurement of 10,000 events per sample was performed with a Gallios flow cytometer (Beckman Coulter). Gating was done based on forward and side scatter properties using CHO RMCE I3 as a negative control. Intracellular HC and LC content was analyzed by FL-6 laser channel using Kaluza Analysis Software (Beckman Coulter). Median fluorescence intensities (MFIs) were compared of three replicates for each antibody variant.

Schwaigerlehner L., Mayrhofer P., Diem M., Steinfellner W., Fenech E., Oostenbrink C, & Kunert R. (2019). Germinality does not necessarily define mAb expression and thermal stability. Applied Microbiology and Biotechnology, 103(18), 7505-7518.

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Multiparametric Analysis of Immune Cells

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Cells were fixed with Foxp3 transcription factor fixation/permeabilization kit (00-5521-00, eBioscience) and blocked using Beriglobin (CSL Behring L, PA, USA). The cells were stained in 100 μL with antibodies and dilutions as shown in Table S2. For biotinylated antibodies, samples were stained with Streptavidin-Alexa Fluor 647 (S32357, Life Technologies). Before acquisition on a FACSVerse (BD Biosciences, San Jose, CA, USA) nuclear stain Hoechst 33342 (H3570, Life Technologies) was added to a concentration of 0.5 μg/ml. Data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For single cell imaging, cells were acquired and analyzed on an ImageStream X Mark II imaging flow cytometer (Amnis, Seattle, WA, USA). CXCL10 levels in undiluted cell culture supernatants were assessed using LEGENDplex reagents (BioLegend, San Diego, CA, USA) according to the manufacturer's instructions.

Bergström B., Lundqvist C., Vasileiadis G.K., Carlsten H., Ekwall O, & Ekwall A.K. (2019). The Rheumatoid Arthritis Risk Gene AIRE Is Induced by Cytokines in Fibroblast-Like Synoviocytes and Augments the Pro-inflammatory Response. Frontiers in Immunology, 10, 1384.

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Confocal Imaging of Lymph Node Cells

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The following Abs/reagents were used for confocal immunofluorescent analysis: biotinylated anti-mouse IgD (11-26/SBA-1, SouthernBiotech), CD4-CF594 (RM4-5, BD biosciences), and streptavidin-Alexa Fluor 647 (Life Technologies) or BCL6-A647(clone K112-91, BD-Pharmingen) and IgM^a-PE (clone DS-1, BD-Pharmingen). Brachial lymph nodes (BLNs) were harvested from recipient mice, fixed for 1 h in 1% paraformaldehyde in PBS on ice and washed with PBS. They were then blocked overnight in 30% sucrose, 0.1% NaN in PBS, embedded in Tissue- Tek optimum cutting temperature compound, snap-frozen in dry ice and ethanol, and stored at −70 °C. Thirty micron cryostat sections were cut from the tissue blocks, affixed to Superfrost Plus microscope slides (Fisher), and stained first with biotinylated anti-IgD Abs and then with anti-CD4 Abs and streptavidin as previously described [22 (link)]. Alternatively they were stained with anti-Bcl6 and anti-IgM^a Abs. Confocal analysis of the sections was performed using Leica SP5 with argon and helium-neon lasers, 2-channel Leica SP spectral fluorescent PMT detector, and a 20× oil-immersion objective with a numerical aperture of 0.7. Images were processed using Imaris (Bitplane).

Turner J.S., Benet Z.L, & Grigorova I.L. (2017). Antigen acquisition enables newly arriving B cells to enter ongoing immunization-induced germinal centers. Journal of immunology (Baltimore, Md. : 1950), 199(4), 1301-1307.

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Immunofluorescent Staining of Frozen Tissue Sections

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After necropsy, organs were frozen in OCT compound (Tissue-Tek^®; Sakura) and transferred in -80°C. Sections of 5 μm were fixed in PFA 4% (Sigma), and washed in PBS-Tween 0.05%. After blocking with Image-IT FX Signal Enhancer (Life Technologies), GL7-FITC antibody (BD) and PNA-biotin (Sigma) were applied at 10 μg/mL overnight at 4°C. Mouse anti-FITC-Alexa Fluor^® 488 antibody (Jackson Immunoresearch) and/ or streptavidin-Alexa Fluor^® 647 (Life Technologies) at 1:200 dilution were used for detection. After counterstaining with 2 ug/ml DAPI (Molecular Probes), slides were washed and mounted in Fluoromount^™ Aqueous Mounting Medium (Sigma) with coverslips. Images were acquired with NanoZoomer 2.0 HT (Hamamatsu) and analyzed with NDP.view2 software (Hamamatsu).

Sordé L., Spindeldreher S., Palmer E, & Karle A. (2017). Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?. PLoS ONE, 12(10), e0186046.

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SARS-CoV-2 S-specific IgG+ Memory B Cell Enrichment

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Starting from freshly isolated PBMCs or upon cells thawing, B cells were enriched by staining with CD19 PE-Cy7 and incubation with anti-PE beads, followed by positive selection using LS columns. Enriched B cells were stained with anti-IgM, anti-IgD, anti-CD14 and anti-IgA, all PE labelled, and prefusion SARS-CoV-2 S with a biotinylated avi tag conjugated to Streptavidin Alexa-Fluor 647 (Life Technologies). SARSCoV-2 S-specific IgG+ memory B cells were sorted by flow cytometry via gating for PE negative and Alexa-Fluor 647 positive cells. Cells were cultured for the screening of positive supernatants. Antibody VH and VL sequences were obtained by RT-PCR and mAbs were expressed as recombinant human Fab fragment or as IgG1 (G1m3 allotype). ExpiCHO cells were transiently transfected with heavy and light chain expression vectors as previously described^{30 (link)}.
Affinity purification was performed on ÄKTA Xpress FPLC (Cytiva) operated by UNICORN software version 5.11 (Build 407) using HiTrap Protein A columns (Cytiva) for full length human and hamster mAbs and CaptureSelect CH1-XL MiniChrom columns (ThermoFisher Scientific) for Fab fragments, using PBS as mobile phase. Buffer exchange to the appropriate formulation buffer was performed with a HiTrap Fast desalting column (Cytiva). The final products were sterilized by filtration through 0.22 μm filters and stored at 4 °C.

Tortorici M.A., Czudnochowski N., Starr T.N., Marzi R., Walls A.C., Zatta F., Bowen J.E., Jaconi S., di iulio J., Wang Z., De Marco A., Zepeda S.K., Pinto D., Liu Z., Beltramello M., Bartha I., Housley M.P., Lempp F.A., Rosen L.E., Dellota E J.r., Kaiser H., Montiel-Ruiz M., Zhou J., Addetia A., Guarino B., Culap K., Sprugasci N., Saliba C., Vetti E., Giacchetto-Sasselli I., Silacci Fregni C., Abdelnabi R., Caroline Foo S.Y., Havenar-Daughton C., Schmid M.A., Benigni F., Cameroni E., Neyts J., Telenti A., Snell G., Virgin H.W., Whelan S.P., Bloom J.D., Corti D., Veesler D, & Pizzuto M.S. (2021). Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody. bioRxiv.

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Flow Cytometry Analysis of HA Binding

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Non-adherent BMDCs were suspended in incubation buffer (PBS + 10% FCS, 0.01% wt/vol sodium azide) and maintained on ice for all incubation steps. Cells were first incubated with Fixable Viability Dye eFluor780 (65-0865; eBioscience) for 15 min, then with TruStain FcX Fc blocker (anti-mouse CD16/CD32 clone 93; BioLegend) for 15 min, before incubation with fluorescently conjugated antibodies for 20 min. Staining for HA was carried out after all other incubations, by fixing the cells in 2% formaldehyde (vol/vol) in PBS for 5 min, then incubating with recombinant biotin-labeled versican G1 domain (bVG1), 3 μg/ml for 40 min followed by streptavidin–Alexa Fluor 647 (S21374; Life Technologies) for 40 min. Cells were counted manually and analyzed using a flow cytometer (LSRII; BD Biosciences) and Flow-Jo software. Compensation was carried out using anti-rat/anti-hamster Ig CompBeads (BD 51-90-9000949) with negative control beads (BD 51-90-9001291), and fluorescence-minus-one controls. As a control for non-specific binding of bVG1, samples were treated with hyaluronidase (HAase, see below) before immunostaining.

Johnson L.A., Banerji S., Lagerholm B.C, & Jackson D.G. (2021). Dendritic cell entry to lymphatic capillaries is orchestrated by CD44 and the hyaluronan glycocalyx. Life Science Alliance, 4(5), e202000908.

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Fluorescent Labeling Reagents Protocol

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Streptavidin-Alexa Fluor 647, streptavidin-Alexa Fluor 555, neutravidin-Oregon Green, and avidin-Alexa Fluor 633 were purchased from Life-Technologies (Invitrogen), dissolved in deionized water (18.2 MΩ·cm at 25 °C) to a concentration of 1 g/L, and stored at −20 °C before use. Pyrrole, NaCl, biotin, Tween-20, and potassium phosphate monobasic and dibasic were obtained from Sigma Aldrich and used as received. BioBeads SM2 were obtained from Bio-Rad. Buffers were freshly prepared in deionized water.

Della Pia E.A., Holm J.V., Lloret N., Le Bon C., Popot J.L., Zoonens M., Nygård J, & Martinez K.L. (2014). A Step Closer to Membrane Protein Multiplexed Nanoarrays Using Biotin-Doped Polypyrrole. ACS Nano, 8(2), 1844-1853.

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Immunohistochemical Analysis of Ebi2-Deficient Thymus

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Ebi2^+/+ and Ebi2^-/- 1-month thymi were embedded in Tissue-Tek OCT (Sakura) and snap frozen. 6-9 μm cryosections were prepared on a Microm HM550 Cryostat (ThermoFisher), stored at -80°C, fixed in 100% acetone at -20°C for 15min and washed 2X in PBS + 0.1% Tween 20. Immunostaining was carried out for 1h at 4°C with the following primary antibodies: anti-Pancytokeratin-FITC (C-11; Sigma-Aldrich), -Keratin5 (rabbit polyclonal antibody; Covance), -CD4-APC (FM4-5; eBiosicence), -CD8-Alexa Fluor 488(53-6.7; eBioscience), CD11c-biotin (N418; BioLegend). The TSA Signal Amplification kit (Perkin Elmer) was used to detect anti-CD11c. Donkey anti-rabbit IgG DyLight 594 (Jackson ImmunoResearch Laboratory) and Streptavidin Alexafluor 647 (Life Technologies) secondary reagents were used for detection. Images were acquired on a DMi8 (Leica) microscope with 10x/0.4 and 20x/0.7 objectives, and processed uniformly using LasX (Leica) software.
To detect autoantibodies in mouse serum, 6-9 μm cryosections from Rag2^-/- kidney sections were prepared. Cryosections were fixed in acetone as described, and incubated overnight with undiluted mouse serum at 4°C. Donkey anti-mouse IgG DyLight594 (Jackson ImmunoResearch Laboratory) was used to detect murine auto-antibodies, followed by a DAPI nuclear counterstain.

Ki S., Thyagarajan H.M., Hu Z., Lancaster J.N, & Ehrlich L.I. (2017). EBI2 contributes to the induction of thymic central tolerance by promoting rapid motility of medullary thymocytes. European journal of immunology, 47(11), 1906-1917.

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