Dextramer

Manufactured by Immudex

Sourced in Denmark

Dextramers are laboratory reagents used for the detection and analysis of antigen-specific T cells. They consist of multiple peptide-major histocompatibility complex (pMHC) molecules bound to a dextran backbone. Dextramers can be used to identify and quantify T cells that recognize specific antigens.

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Lab products found in correlation

Anti cd44 bv510, by BioLegend (2 mentions) Anti cd8 apc h7, by BD (2 mentions) Facsaria, by BD (2 mentions) Pentamers, by ProImmune (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Pacific blue anti mouse cd3, by BioLegend (1 mentions) Facsdiva software, by BD (1 mentions) Fix perm buffer, by Thermo Fisher Scientific (1 mentions) Tcs sp8, by Leica camera (1 mentions) Accuri c6, by BD (1 mentions) Fixable viability dye, by Thermo Fisher Scientific (1 mentions) Mouse fc block, by BD (1 mentions) Anti cd3 percp cy5, by Cytek Biosciences (1 mentions) Anti cd43 pe cy7, by BioLegend (1 mentions) Streptavidin sa, by Solarbio (1 mentions) M13mp18 dna, by New England Biolabs (1 mentions) Oligonucleotide, by Sangon (1 mentions) Cd8 microbead, by Miltenyi Biotec (1 mentions) Anti perforin, by Mabtech (1 mentions) Anti pd 1 eh12.2h7, by BioLegend (1 mentions)

15 protocols using dextramer

HLA/Peptide Multimer Antigen Presentation

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PE-conjugated HLA/peptide multimers were dextramers (Immudex,
Copenhagen, Denmark), pentamers (Pro-immune, Oxford, UK) or tetramers (NIH
tetramer facility, Atlanta, GA) and presented published HDV epitopes.^{6 –8}

Kefalakes H., Horgan X.J., Jung M.K., Amanakis G., Kapuria D., Bolte F.J., Kleiner D.E., Koh C., Heller T, & Rehermann B. (2021). Liver-resident bystander CD8+ T cells contribute to liver disease pathogenesis in chronic hepatitis D virus infection. Gastroenterology, 161(5), 1567-1583.e9.

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Phenotyping Antigen-Specific CD8+ T Cells

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Single-cell suspensions of splenocytes (1 x 10⁶ cells/well) were first stained for 45 min at 4°C with H-2Kb (SIINFEKL) dextramers conjugated to phycoerythrin (PE) (Immudex, Copenhagen, Denmark). Cells were then incubated with Pacific Blue^™ anti-mouse CD3 (Biolegend, San Diego, CA, US), anti-mouse CD8a APC-eFluor^® 780 (eBioscience, San Diego, CA, US) and anti-mouse CD62L (L-Selectin) Alexa Fluor^® 700 (eBioscience) antibodies for 30 min at 4°C. After fixation and permeabilization using Fix/Perm buffer (eBioscience), cells were intracellularly stained with anti-human/mouse T-bet PE-Cyanine7 (eBioscience) and Alexa Fluor^® 647 anti-human/mouse Granzyme B (Biolegend) antibodies for 1 h at 4°C. Flow cytometry analysis was performed using a BD LSRII flow cytometer and BD FACSDIVA software (Becton Dickinson, NJ, US). The results were analyzed using FLOWJO software.

Carpentier A.F., Geinguenaud F., Tran T., Sejalon F., Martin A., Motte L., Tartour E, & Banissi C. (2017). Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses. PLoS ONE, 12(7), e0181403.

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CD1d-Dextramer Lipid-Ligand Loading

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A standard protocol recommended by the company including some modifications according to the stability of the lipid-ligand was used to load the CD1d/unloaded Dextramers®(Immudex TF1037.01) with the lipid-ligand of Pru p 3. The lipid was dissolved in methanol at 1 mg/ml and dissolved completely at room temperature. Dextramer with the lipid was always kept at 2–8°C in the dark until its use with cultured cells. The binding to experimental cells was performed at 37°C for 10 min, and then analysed by flow cytometry (BD-Accuri C6, BD-Biosciences, USA) or confocal microscopy (Leica TCS-SP8)

Tordesillas L., Cubells-Baeza N., Gómez-Casado C., Berin C., Esteban V., Barcik W., O’Mahony L., Ramirez C., Pacios L.F., Garrido-Arandia M, & Díaz-Perales A. (2017). Mechanisms underlying induction of allergic sensitization by Pru p 3. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 47(11), 1398-1408.

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Peptide-based Epitope Mapping for HEV

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All peptides used in the study were synthesized by ProImmune, UK. Peptide sequences specific to HEV are of genotype 3, based on data from GenBank accession number AF455784. Peptides were dissolved in Dimethyl sulfoxide (DMSO) to yield stock solutions of 60 mg/mL, which were further diluted with HBSS to be used in cell culture.
Dextramers bearing the selected epitopes were synthesized by Immudex, Denmark. All Dextramers were specific for HLA-A^*02:01 allele, and conjugated with PE fluorochrome (except HEV-1527 Dextramer, which was conjugated with APC fluorochrome). In Dextramer dilution experiment, various dilutions were prepared by using FACS buffer as diluent to yield the dilution factors as indicated in Figure 4.

Soon C.F., Zhang S., Suneetha P.V., Antunes D.A., Manns M.P., Raha S., Schultze-Florey C., Prinz I., Wedemeyer H., Sällberg Chen M, & Cornberg M. (2019). Hepatitis E Virus (HEV)-Specific T Cell Receptor Cross-Recognition: Implications for Immunotherapy. Frontiers in Immunology, 10, 2076.

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Characterization of HSV-specific CD8+ T Cells

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Characterization of the number and phenotype of HSV-specific CD8⁺ T cells specific to the SSI peptide was performed by flow cytometry using dextramers (Immudex, Copenhagen, Denmark), as previously described [16 (link)]. The following antibodies were used: PerCP-Cy5.5 anti-CD3 (TONBO Biosciences, Societa Italiana Chimici Rome, Italy); APC anti-CD62L (Immunotools, Friesoythe, Germany); BV510 anti-CD44 (Biolegend, Campoverde S.r.l. Milano, Italy) and APC-H7 anti-CD8 (Becton Dickinson Milano, Italy). Samples were acquired on FACS Aria flow cytometer (BD) within 2 h of fixation. Flow cytometry data were analyzed using FlowJo (version 9.5.3; Tree Star Inc., Ashland, OR, USA).
Sera for antibody determinations were collected, stored and assessed by using the ELISA test for the presence and titers of anti-HSV IgG, as previously described [23 (link)].

Nicoli F., Gallerani E., Sicurella M., Pacifico S., Cafaro A., Ensoli B., Marconi P., Caputo A, & Gavioli R. (2020). The Tat Protein of HIV-1 Prevents the Loss of HSV-Specific Memory Adaptive Responses and Favors the Control of Viral Reactivation. Vaccines, 8(2), 274.

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Neoantigen Peptide Screening Protocol

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SIINFEKL peptide (VACSIN) and CpG(tlr-1826-1) were from InvivoGen (San Diego, CA). Neoantigen peptides were purchased from LifeTein (Somerset, NJ) and BioSynthesis (Lewisville, TX). Dextramers were purchased from Immudex (Copenhagen, Denmark).

Coder B., Pryshchep O., Kelkar D., Filippova E.V., Ju X., Balli D., Mottershead C., Ramos K., Thambi N., Cheng Z., Lugt B.V., Lesch J., Liu X., DeVoss J., Cooke K.S., Liu S., Zhan J., Mitchell P., Villarreal D.O., Hayes S.M., Johnston J.A., Petit R.G., Phee H., & Princiotta M.F. (2020). Listeria monocytogenes personalized cancer vaccines drive therapeutic immune responses to cancer derived neoantigens.

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Dextramer Staining of Splenocytes and Tumor Cells

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For dextramer staining, a total of 1 x 10 6 splenocytes or 2 x 10 6 tumor cells were washed twice with FACS buffer (PBS, 2% FBS, 2mM EDTA), stained with dextramers (Immudex) for 10 minutes at room temperature. Without additional washing, an antibody cocktail containing Fc block (14-0161-86, eBioscience) BV buffer (566349, BD Bioscience) were added. Cells were incubated at 4 o C for 20 minutes, washed in FACS buffer, stained with fixable viability dye (65-0866-14, eBioscience) for 10 minutes at room temperature, washed with FACS buffer and flow cytometry was performed within 2 hours of final wash.

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Optimized pMHC Multimer Staining Protocol

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The pMHC multimer staining method was adapted from that of Dolton et al. (38) . Cells were incubated with a protein kinase inhibitor, 50nM dasatinib, for 30 minutes at 37°C. PE-or APC-conjugated tetramers or Dextramers were centrifuged at 16,000 g for 1 minute to remove aggregates. The cells were stained with 0.5μg of pMHC tetramer or pMHC dextramer at 6.4nM in 50μL (unless stated otherwise) for 30 minutes on ice or for one hour at room temperature. Following pMHC multimer staining, the cells were washed with cold staining buffer twice.
Samples were incubated with mouse Fc Block (BD Biosciences, catalogue# 553141) for 10 minutes at 4°C and either or both mouse anti-PE and anti-APC were added at 0.5µg/100µL depending on the pMHC multimer conjugates used. The cells were washed and incubated with a cocktail of antibodies against surface markers for 30 minutes on ice. Viability staining, acquisition and analysis were performed as above. Dextramers were purchased from Immudex. QuickSwitch Custom Tetramer Kits (MBL International) were utilised to generate multiple tetramers with selected peptides in order to screen an array of pMHC epitopes. Quantitation of peptide exchange with selected peptides was performed according to the manufacturer's protocol.

Son E.T., Faridi P., Paul-Heng M., Leong M., English K., Ramarathinam S.H., Braun A., Dudek N.L., Alexander I.E., Lisowski L., Bertolino P., Bowen D.G., Purcell A.W., Mifsud N.A., & Sharland A. (2020). The self-peptide repertoire plays a critical role in transplant tolerance induction.

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Phenotyping HSV-specific CD8+ T Cells

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Characterization of number and phenotype of HSV-specific CD8 + T cells was done by flow cytometry using dextramers (Immudex, Copenhagen, Denmark) to identify CD8 + T cells specific for the HSV1 Kb-restricted SSI and QTF epitopes as previously described [15] . The following antibodies were used: anti-CD3 PerCP-Cy5.5, anti-KLRG1 APC and anti-CD127 PE-Cy7 (TONBO Biosciences, Società Italiana Chimici Rome, Italy), anti-CD62L APC (Immunotools, Friesoythe, Germany), anti-CD43 PE-Cy7 (activated isoform) and anti-CD44 BV510 (Biolegend, Campoverde S.r.l. Milano, Italy), anti-CD103 BV510, anti-CD27 V450 and anti-CD8 APC-H7 (Becton Dickinson Milano, Italy). Samples were acquired on FACS Aria flow cytometer (Becton Dickinson). Flow cytometry data were analyzed using FlowJo (version 9.5.3; Tree Star Inc., Ashland, USA). Memory precursor effector cells (MPEC) were defined as KLRG1 -CD127 + , shortlived effector cells (SLEC) as KLRG1 + CD127 -, central memory T cells (Tcm) as CD44 + CD62L + and effector memory T cells (Tem) as CD44 + CD62L -.

Nicoli F., Gallerani E., Skarlis C., Sicurella M., Cafaro A., Ensoli B., Caputo A., Marconi P.C, & Gavioli R. (2016). Systemic immunodominant CD8 responses with an effector-like phenotype are induced by intravaginal immunization with attenuated HSV vectors expressing HIV Tat and mediate protection against HSV infection. Vaccine, 34(19).

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Molecular Immunology Reagent Preparation

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M13mp18 DNA was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Oligonucleotides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) with standard desalting and purified with HPLC, and used without further purification. Information on the DNA oligonucleotide sequences are provided in Supplementary Data 1. Streptavidin (SA) and PE-SA were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Biotin-labeled peptide–MHC monomers (H-2K^b-OVA_257-264, H-2K^d-InsB, HLA-A*0201 CMV pp65, etc.) and tetramers were provided by MBL Biotech Co., Ltd (Japan). Dextramer was provided by Immudex (Copenhagen, Denmark). Fluorescent anti-mouse/human antibodies used for flow cytometry analysis were purchased from BioLegend. MTT Cell Proliferation and Cytotoxicity Assay Kit (cat. 606334) were provided by Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals were obtained from Sinopharm and Sigma-Aldrich.

Sun Y., Yan L., Sun J., Xiao M., Lai W., Song G., Li L., Fan C, & Pei H. (2022). Nanoscale organization of two-dimensional multimeric pMHC reagents with DNA origami for CD8+ T cell detection. Nature Communications, 13, 3916.

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