Anti cd71

Manufactured by BioLegend

Sourced in United States

Anti-CD71 is an antibody that binds to the CD71 (transferrin receptor) antigen, which is expressed on the surface of various cell types involved in cellular metabolism and proliferation. It is commonly used in flow cytometry and other applications to detect and quantify CD71-expressing cells.

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11 protocols using anti cd71

HPV16 Peptide-Mediated Immune Response

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On D0, PBMCs from 6 healthy women donors (from 25 to 35 years old) were incubated at the density of 200,000 cells/well in 96-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium (Panbiotech, P04-17500), added with 2% human decomplemented serum, 1 mM non-essential amino acids, 1 mM pyruvate, 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, in the presence of HPV16_(L1) peptide mix at the final dilution of 1/450 v/v. On D2, either the Veh., MIM-1; -2; -3; -4 or -5 were added to the medium and incubated for the next 72 h at the final sucrose–lactose concentration of 11 mM. Controls with either HPV16_(L1) or IL-2 (20 ng/mL) were also run in parallel. On D5, the cells were harvested, saturated with FcBlock solution (BD, 564220), immune-stained with fluorescent anti-CD3, anti-CD4, anti-CD8, anti-CD71, anti-CD95, anti-CD28 and anti-HLA-DR antibodies (all purchased from BioLegend), fixed, and analyzed by flow cytometry. The discrimination of the cell sub-populations is as follows: CD4⁺: CD3^high; CD4^high; CD8^low; SSC^low, CD8⁺: CD3^high, CD4^low, CD8^high, SSC^low, CD4⁻/CD8⁻: CD3^high; CD4^low; CD8^low; SSC^low. The supernatants (SNs) were retrieved and the cytokine levels of IFN-γ, IL-6, and interferon- γ inducible protein (IP-10) were assessed by enzyme-linked immunosorbent assay (ELISA).

Jacques C., Marchand F., Chatelais M., Albinet V., Coustal C, & Floris I. (2024). The Micro-Immunotherapy Medicine 2LPAPI® Displays Immune-Modulatory Effects in a Model of Human Papillomavirus Type-16 L1-Protein Capsid-Treated Human Peripheral Blood Mononuclear Cells and Antiproliferative Effects in a Model of Cervical Cancer Cells. Cancers, 16(7), 1421.

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Monoclonal Antibodies for Cell Surface Marker Analysis

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Rat monoclonal anti‐SSEA3 (Clone MC‐631) along with the following mouse monoclonal antibodies were used: anti‐SSEA3, anti‐SSEA4 (Clone MC‐813–70), anti‐CD34 (Clone V MA27), anti‐CD41 (Integrin GPIIb, Clone IV P38), anti‐CD235ab (Glycophorin A/B, Clone VII 70299), anti‐CD235a (Glycophorin A, Clone VII 70312), anti‐CD71 (transferrin receptor, Clone A015) (Biolegend, San Diego, CA); anti‐CD238 (Kell, Clone BRIC 203), anti‐CD234 (Duffy, Clone 2C3) (BD Biosciences, San Jose, CA); anti‐RhAG (Clone LA1818) (kindly provided by Prof Ellen van der Schoot, Sanquin, Amsterdam, Netherlands); anti‐RhD/CE (Clone BRIC69, Fisher Scientific); anti‐U (Glycophorin B, Human plasma, Versiti‐Wisconsin); anti‐CD233 (BAND3, Clone BRIC6, American Research Products, Palos Verdes Estates, CA); anti‐CD173 (H antigen, Clone BRIC 231, MyBioSource, San Diego, CA). Secondary antibodies used were as follows: monoclonal APC‐conjugated Rat anti‐mouse IgG₁ (Clone RMG1‐1, Biolegend) and polyclonal APC‐conjugated goat anti‐human IgG (Fab) (RRID: AB_2337691, Jackson Laboratories, Bar Harbor, ME).

Pandey P., Zhang N., Curtis B.R., Newman P.J, & Denomme G.A. (2021). Generation of ‘designer erythroblasts’ lacking one or more blood group systems from CRISPR/Cas9 gene‐edited human‐induced pluripotent stem cells. Journal of Cellular and Molecular Medicine, 25(19), 9340-9349.

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Murine Immune Cell Profiling

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A single cell suspension from thymi and spleens of 4–6 week-old mice was obtained using a Dounce homogenizer. Cells were first preincubated for 10 min on ice with Fc receptor-blocking anti-CD16/32 (clone 93) antibody in PBS-1% biotin-free BSA solution and then stained for 20 min with specific primary antibodies. Biotinylated primary antibodies were revealed with streptavidin conjugated fluorescent dyes (SAv-PE/Cy7, SAv-APC, SAv-APC/Cy7). Labeled cells were analyzed on BD FACSCantoII (BD Biosciences) or CytoFLEX (Beckman Coulter) flow cytometers and data analysis was performed using FlowJo software (FlowJo, LLC). Clones of monoclonal antibodies were: anti-CCR6 (29–2L17), anti-CD3ε (145–2C11), anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.7), anti-CD11c (N418), CD24 (M1/69), anti-CD25 (PC61), anti-CD27 (LG.3A10), anti-CD28 (E18), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 (RI7217), anti-CD73 (TY/11.8), anti-CD117 (c-Kit, 2B8) anti-B220 (RA3–6B2), anti-CD11b (Mac1, M1/70), anti-γδT-cell (GL3), hamster IgG-PE/Cy7 (HTK888), anti-Ly6G/Ly6C (GR1, RB6–8C5), anti-Nk1.1 (PK136), anti-Tcrβ (H57–597), anti-Tcrβ(Vα2) (B20.1), Ter-119 (all from Biolegend). Splenocytes were analyzed as previously described (18 (link)).

Zikmund T., Kokavec J., Turkova T., Savvulidi F., Paszekova H., Vodenkova S., Sedlacek R., Skoultchi A.I, & Stopka T. (2019). ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β-selection.. Journal of immunology (Baltimore, Md. : 1950), 202(12), 3434-3446.

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Quantifying Immune Cell Phenotypes

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We performed cell surface staining with fluorescence-conjugated antibodies in 2% FBS–PBS. We obtained fluorochrome-conjugated anti-CD11b, anti-F4/80, anti-CD71, and anti-CD98 from Biolegend. We identified cell death using a violet Live/Dead kit (Invitrogen). We performed intracellular staining for Alexa Fluor 647-conjugated p-Akt (Ser473) and p-4EBP1 (Thr37/46) (Cell Signaling Technology) using BD Biosciences Cytofix/Cytoperm and Perm/Wash solutions. We collected data using a BD FACSAria II and analyzed them with FlowJo (Treestar).

Xie D.L., Zheng M.M., Zheng Y., Gao H., Zhang J., Zhang T., Guo J.C., Yang X.F., Zhong X.P, & Lou Y.L. (2017). Vibrio vulnificus induces mTOR activation and inflammatory responses in macrophages. PLoS ONE, 12(7), e0181454.

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Isolation of Murine Erythroid Progenitor and T Cell Populations

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Splenocytes were collected by mechanical disruption. Cell suspensions were passed through 70-μm cell strainers and then washed and resuspended in staining buffer. For the isolation of spleen CD45⁺Ter119⁺CD71⁺cells(CD45⁺EPC), cells were stained with anti-CD45 monoclonal antibody (mAb),anti-Ter119 (mAb), and anti-CD71 (both from Biolegend). Then, CD45⁺EPC were sorted using the BD FACSAsia II Special Order System. For spleen CD8⁺ T cell isolation, CD8⁺T lymphocytes were isolated from spleens of wild type mice using T-cell enrichment columns (Stem Cell). For all sorted samples, a purity of greater than 95% was achieved.

Fan X., Peng H., Wang X., Sun Y., Dong Y., Zhou J., Chen J, & Huang S. (2024). Tumor-associated CD8+T cell tolerance induced by erythroid progenitor cells. Frontiers in Immunology, 15, 1381919.

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Characterization of Neural Derived Progenitor Cells

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nDPSC were harvested by trypsin/EDTA treatment for 5 min at 37 °C. After inactivation of trypsin and centrifugation (5 min at 300 g), 100 µl of the cell suspension (10⁵ cells) was incubated for 30 min at room temperature in the dark with the following fluorochrome-conjugated antibodies: anti-CD10 (eBiosciences; Clone: CB-CALLA), anti-CD13 (eBiosciences; Clone: WM-15), anti-CD29 (Chemicon, Clone: TDM29), anti-CD34 (Biolegend; Clone: 4H11), anti-CD44 (Caltag; Clone: MEM-85), anti-CD45 (Caltag; Clone: HI30), anti-CD71 (Biolegend; Clone: MEM-75), anti-CD73 (BD Pharming; Clone: AD2), anti-CD90 (Chemicon; Clone: F15-42-1), anti-CD105 (Caltag; Clone: SN6), anti-CD146 (Millipore; Clone: P1H12), anti-CD166 (Beckman; Clone: 3A6), anti-CD222 (eBiosciences; Clone: MEM-238), anti-CD271 (Biolegend; Clone: ME20.4), anti-CXCR4 (Caltag; Clone: 12G5), anti-INF-beta (INFsource; Clone: MMHB-3) and anti-HLA-DR (Caltag; Clone: Tu36). After washing with PBS, at least 10,000 events were acquired using a Beckman Coulter FC500 flow cytometer. Instrument settings and gating strategies were established using isotype controls, and data were analysed using Kaluza software (Beckman Coulter). All experiments were carried out in duplicates.

Pisal R.V., Suchanek J., Siller R., Soukup T., Hrebikova H., Bezrouk A., Kunke D., Micuda S., Filip S., Sullivan G, & Mokry J. (2018). Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth. Scientific Reports, 8, 6168.

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Flow Cytometry Analysis of PBMCs

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Fresh PBMCs were used for each flow cytometry analysis. Cells were suspended in phosphate-buffered saline (PBS) and incubated with antibody cocktails in the dark at room temperature for 15 min. After incubation, the cells were washed and resuspended in PBS and then analyzed. Data were acquired using an EC-800 (Sony Biotechnology Inc., Champaign, IL, USA) and analyzed with the Kaluza software program (Beckman Coulter, Brea, CA, USA). Forward scatter and side scatter gates were set to capture the cell population of interest, including monocytes and lymphocytes. We excluded the debris fractions to eliminate aberrant binding events due to dead cells as other groups did^{34 (link),51 (link)}. The following antibodies were used: anti-GPA (17-9987-42; eBiosciences, San Diego, CA, USA), anti-CD36 (IM0766U; Beckman Coulter), anti-CD45 (2120080; Sony Biotechnology Inc.), and anti-CD71 (334106; Biolegend, San Diego, CA, USA).

Kanemasa H., Ishimura M., Eguchi K., Tanaka T., Nanishi E., Shiraishi A., Goto M., Motomura Y, & Ohga S. (2021). The immunoregulatory function of peripheral blood CD71+ erythroid cells in systemic-onset juvenile idiopathic arthritis. Scientific Reports, 11, 14396.

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Macrophage Polarization and JUNV Infection

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MEM, RPMI, and FBS were purchased from Invitrogen (Buenos Aires, Argentina). rM-CSF was acquired from R&D Systems (Minneapolis, MN, USA). Anti JUNV antibodies were obtained from BEI resources, USA. Anti CD71, CD14, CD86, CD80, HLA-DR, CD11b, CD11c, CD64, CD163, CD206 were obtained from BioLegend (San Diego, CA, USA). Anti-human APC-MERTK (mouse IgG1), Biotin-AXL (goat IgG) and isotype controls were obtained from R&D Systems (Minneapolis, MN, USA). Anti-TYRO3 (rabbit IgG) was obtained from Novus Biological (Littleton, CO, USA). DAPI was purchased from Invitrogen (Buenos Aires, Argentina). ELISA kits (Ready-SET-Go kits) for TNF-α, IL-1β, IL-6, IL-10, and IL-12p70 were obtained from eBioscience, Fisher scientific, Pittsburgh, PA, USA. Cytofix/Cytoperm kit was purchased from BD Bioscience (San Diego, CA, USA).

Ferrer M.F., Thomas P., López Ortiz A.O., Errasti A.E., Charo N., Romanowski V., Gorgojo J., Rodriguez M.E., Carrera Silva E.A, & Gómez R.M. (2019). Junin Virus Triggers Macrophage Activation and Modulates Polarization According to Viral Strain Pathogenicity. Frontiers in Immunology, 10, 2499.

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Murine CD4+ T Cell Activation

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For in vitro experiments, primary murine naïve CD4 T cells were isolated from the spleens and LNs of female control C57BL/6 (JAX, #000664) or age-matched lupus-prone SLE1.2.3 mice (JAX, #007228) using the CD4⁺CD62L⁺ T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec). CD62L-positive selection was routinely ~98% in purity, as determined by flow cytometry. Cell cultures were activated in the presence of either anti-CD71 (BioLegend, 113821) or isotype control (IgG2a; BioLegend, 400543) at 2 μg/ml, and downstream experiments were performed between days 3 and 6 after activation. For in vivo anti-CD71 experiments, female control or age-matched SLE1.2.3 mice were treated with 200 μg of CD71-blocking antibody (clone R17 217.1.3/TIB-219) or an isotype control antibody starting at 4 to 5 months of age (Bio X Cell). Treatment was administered twice a week via intraperitoneal injection for 4 weeks.

Voss K., Sewell A.E., Krystofiak E.S., Gibson-Corley K.N., Young A.C., Basham J.H., Sugiura A., Arner E.N., Beavers W.N., Kunkle D.E., Dickson M.E., Needle G.A., Skaar E.P., Rathmell W.K., Ormseth M.J., Major A.S, & Rathmell J.C. (2023). Elevated transferrin receptor impairs T cell metabolism and function in systemic lupus erythematosus. Science immunology, 8(79), eabq0178.

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Cell Proliferation Analysis by Flow Cytometry

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To measure cell proliferation, cells were stained with a CellTrace Violet Cell Proliferation kit (Thermo Scientific) prior to culturing. Flow cytometry staining was undertaken in 96-well round-bottom plates with a minimum of 200,000 cells per well. To distinguish live cells, the cells were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Thermo Scientific). For surface staining, the cells were incubated in Facs buffer (2.5% FBS and 0.05% sodium azide in PBS) with labeled antibodies, all Thermo Scientific unless otherwise indicated: anti-CD8β, anti-CD25, anti-CD69 (BioLegend), anti-CD71 (BioLegend) and anti-CD98. Intranuclear staining was done using the eBioScience Foxp3/Transcription Factor Staining kit (Thermo Scientific) and anti-Irf4, anti-T-bet, and anti-c-Myc (Cell Signaling) antibodies. Flow cytometry was performed on a MacsQuant Analyzer 10. Flow cytometry data were analyzed using FlowJo 10 software. Flow cytometry data were generated within the Flow Cytometry and Cell Sorting Facility in Ashworth, King's Buildings at the University of Edinburgh.

Toivakka M., Gordon K., Kumar S., Bermudez-Barrientos J.R., Abreu-Goodger C., Zamoyska R, & Buck A.H. (2024). miR-7 is recruited to the high molecular weight RNA-induced silencing complex in CD8+ T cells upon activation and suppresses IL-2 signaling. RNA, 30(1), 26-36.

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