Cd45ro

Manufactured by BioLegend

Sourced in United States

CD45RO is a cell surface marker that identifies memory T cells. It is a high molecular weight isoform of the CD45 protein.

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Lab products found in correlation

Cd45ra, by BioLegend (8 mentions) Cd62l, by BioLegend (7 mentions) Tim 3, by BioLegend (5 mentions) T bet, by BioLegend (4 mentions) Cd107a, by BioLegend (4 mentions) Ifn γ, by BD (4 mentions) Klrg1, by BioLegend (4 mentions) Cd137, by BioLegend (4 mentions) Tnf α, by BioLegend (3 mentions) Lag 3, by BD (3 mentions) Cd11c, by BioLegend (3 mentions) Cd127, by BioLegend (3 mentions) Ifn γ, by BioLegend (3 mentions) Cd127, by BD (3 mentions) Cd49d l25, by BD (2 mentions) Cd103, by BD (2 mentions) Unlabeled cd28 l293, by BD (2 mentions) Eomesodermin wd1928 pe efluor610, by Thermo Fisher Scientific (2 mentions) S1pr1 sw4gypp efluor660, by Thermo Fisher Scientific (2 mentions) Mip 1β, by BD (2 mentions)

25 protocols using cd45ro

Detecting αvβ6 and CAR Expression

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Expression of αvβ6 was detected using the 6.3G9
antibody,46 (link) followed by goat
anti-mouse Ig-PE (Dako, Ely, UK) and was expressed as percentage positivity and/
or geometric mean fluorescence intensity. Cells stained with secondary antibody
only served as a negative control. Expression of CARs was detected using
supernatant derived from the 9e10 hybridoma (ECACC), which binds to residues
410-419 of human c-myc, followed by goat anti-mouse Ig-PE. Untransduced T-cells
acted as a negative control. CD8 expression was detected using PE-conjugated
PNIM0452 (Immunotech, Marseille, France). Phenotypic analysis of T-cells was
performed using anti-CCR7 (R&D systems FAB197F) and CD45RO (Biolegend
304210) antibodies. In some assays, populations were gated on CAR⁺cells detected using an antibody against CD124 (BD Pharmingen, 552178). To
assess integrin specificity of the A20 peptide, biotinylated peptide (Genscript,
Hong Kong Ltd) was diluted in PBS supplemented with MgCl₂ and NaCl
(Sigma D8662) and incubated with cells on ice for 20 minutes before being
stained with streptavadin-PE (LifeTech S866) for a further 20 minutes on ice.
Flow cytometry was performed using a FACSCalibur cytometer with CellQuest Pro
software or Fortessa cytometer with FACSDiva software.

Whilding L.M., Parente-Pereira A.C., Zabinski T., Davies D.M., Petrovic R.M., Kao Y.V., Saxena S.A., Romain A., Costa-Guerra J.A., Violette S., Itamochi H., Ghaem-Maghami S., Vallath S., Marshall J.F, & Maher J. (2017). Targeting of Aberrant αvβ6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells. Molecular Therapy, 25(1), 259-273.

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Multiparametric Flow Cytometry Assay

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The following monoclonal antibodies were used in flow cytometry: CD8 (SK1: APC-H7), CCR7 (3D12: PE-Cy7), CD69 (L78: PE), IFN-γ (B27: PE-Cy7), MIP-1β (D21-1351: PerCP-Cy5.5), from BD Biosciences (San Jose, CA); CD103 (Ber-ACT8: Ax488), TNF-α (MAb11: BV605), IL-2 (MQ1-17H12: BV650), CD107a (H4A3: BV711), CD4 (RPA-TA: BV570, BV605), CD45RO (UCHL1: BV785), CD27 (O323: BV650), CD3 (UCHT-1: Ax700), and T-bet (4B10: BV711) from Biolegend (San Diego, CA). Unlabeled CD28 (L293), and CD49d (L25) were from BD Pharmingen (San Diego, CA). Eomesodermin (WD1928: PE-eFluor610) and S1PR1 (SW4GYPP: eFluor660) were from ThermoFisher (Waltham, MA). The HIV-1 Gag peptide pool (p55, HXB2 sequence) consisted of 15-mers with an 11 amino acid overlap (BD Biosciences). Staphylococcal enterotoxin B (SEB) was from Sigma Aldrich.

Kiniry B.E., Li S., Ganesh A., Hunt P.W., Somsouk M., Skinner P.J., Deeks S.G, & Shacklett B.L. (2017). Detection of HIV-1-Specific Gastrointestinal Tissue Resident CD8+ T-cells in Chronic Infection. Mucosal immunology, 11(3), 909-920.

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Immune Reconstitution in Humanized Mouse Model

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Human immune cell reconstitution in hu-mice was analyzed by flow cytometry (FCM) using various combinations of the following monoclonal antibodies: anti-human CD45, CD3, CD4, CD8, CD45RA, CD45RO, CD69, CCR7, CD31 (all purchased from Biolegend, San Diego, CA, USA); and anti-mouse CD45 (BD Pharmingen) and Ter119 (Biolegend, San Diego, CA, USA). Peripheral blood was collected from tail vein into heparinized tubes, and mononuclear cells were purified by density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). FCM analysis was performed on a FACS Fortessa (BD Biosciences). Dead cells were excluded from the analysis by gating out lower forward scatter and high propidium iodide–retaining cells. Data analysis was performed using FlowJo 10.3 software.

Tong Q.Y., Zhang J.C., Guo J.L., Li Y., Yao L.Y., Wang X., Yang Y.G, & Sun L.G. (2020). Human Thymic Involution and Aging in Humanized Mice. Frontiers in Immunology, 11, 1399.

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Comprehensive Antibody Panel for Mouse and Human T Cell Phenotyping

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For mouse studies, the following antibodies were purchased: from BioLegend: 2B4 (m2B4), BCL-2 (BCL/10C4), CD101 (Moushi101), CD11c (N418), CD127 (A7R34), CD19 (6D5), CD25 (PC61.5), CD3 (145–2C11), CD38 (90), CD39 (Duha59), CD40 (3/23), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD70 (FR70), CD80 (16–10A1), CD86 (GL-1), CD90.1 (OX-7 and HIS51), CD90.2 (30-H12 and 53–2.1), CXCR5 (L138D7), Eomes (Dan11mag), GZMB (GB11), IFNγ (XMG1.2), IL-2 (JES6–5H4), KLRG1 (2F1), LAG-3 (C9B7W), MHC I-A/I-E (M5/114.15.2), PD-1 (RMP1–30), T-bet (4B10), TIM-3 (RMT3–23), TNF (MP6-XT22), and 7-amino-actinomycin (7-AAD); from BD Biosciences: annexin V, CD95 (Jo2), Ki67 (B56), Vb7 (TR310); BCL-xL (H-5; Santa Cruz Biotechnology); BIM (C34C5; Cell Signaling Technology), CD8 (53–6.7; eBioscience), CTLA-4 (UC10–410-11; Tonbo Biosciences), TCF-1 (C63D9; Cell Signaling Technology), TIGIT (GIGD7; eBioscience).
For human studies, the following antibodies were purchased: CD39 (A1; BioLegend), CD45RA (HI100; BioLegend), CD45RO (UCHL1; BioLegend), CD8 (RPA-T8; BioLegend), LAG-3 (17B4; Enzo Life Sciences), PD-1 (EH12.1; BD Biosciences) and TIM-3 (F38–2E2; BioLegend).
For flow cytometric detection and analysis of mouse and human TOX, anti-human/mouse TOX antibody clone REA473 was used (Miltenyi Biotec); antibody clone REA293 was used as TOX isotype (Miltenyi Biotec).

Scott A.C., Dündar F., Zumbo P., Chandran S.S., Klebanoff C.A., Shakiba M., Trivedi P., Menocal L., Appleby H., Camara S., Zamarin D., Walther T., Snyder A., Femia M.R., Comen E.A., Wen H.Y., Hellmann M.D., Anandasabapathy N., Liu Y., Altorki N.K., Lauer P., Levy O., Glickman M.S., Kaye J., Betel D., Philip M, & Schietinger A. (2019). TOX is a critical regulator of tumour-specific T cell differentiation. Nature, 571(7764), 270-274.

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Mitochondrial Function in T Cell Activation

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PBMCs were plated in a 96-well flat-bottom plate and cultured in RPMI-1640 supplemented with 10% FBS at a density of 2 × 10⁶ cells per well. Cells were stimulated with coated anti-CD3 (Clone: OKT3; 2 µg/mL, Biolegend, San Diego, CA, USA) or PBS control and measured at 4 h and 24 h time points. For the final one hour of stimulation MitoTracker Green (100 nm, Thermo Fisher Scientific, Waltham, MA, USA) and TMRM (25 nM, Sigma-Aldrich, St. Louis, MI, USA) were added to the wells at 37 °C/5% CO₂. After culture, cells were stained with cell surface markers anti-CD3, -CD4, -CD27, -CD28, -CD45RO and -CD45RA (BioLegend, San Diego, CA, USA). Cell viability was assessed using Live/Dead Aqua (ThermoFisher, Waltham, MA, USA). Cells were immediately analysed using the BD LSRFortessa^TM X-20 cell analyser (BD Biosciences, Franklin Lakes, NJ, USA). Separately stained cells were used to define compensation parameters.

Strickland M., Lee S., Neo S.Y., Balachander A., Low I., Mustafah S., Goh W.I., Wright G.D., Larbi A, & Pender S.L. (2023). Mitochondrial Dysfunction in CD4+ T Effector Memory RA+ Cells. Biology, 12(4), 597.

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Multiparameter Immune Cell Profiling

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Blister cells were suspended in 100 μl of cell staining buffer (PBS with 5% FCS, 0.1% sodium azide) were incubated for 30 mins on ice with following antibodies: CD3: FITC, CD14: BV605, CD16: APC, CD19: FITC, CD56: FITC, CD62L: PE-Cy7, CD163: BV421, HLA-DR: BV510, CD45RO: BV785, CCR7: BV711 (all antibodies were obtained from Biolegend). Stained cell sample was washed to remove excess antibody and then fixed in 1% paraformaldehyde. Fixed sample was acquired on BD LSR Fortessa within 4h. Flow cytometry data was analysed by Flowjo software (Treestar Inc.). Flow cytometric gating strategy employed to identify cell populations is as described in reference [13 (link)].

Motwani M.P., Newson J., Kwong S., Richard-Loendt A., Colas R., Dalli J, & Gilroy D.W. (2017). Prolonged immune alteration following resolution of acute inflammation in humans. PLoS ONE, 12(10), e0186964.

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Multiparameter Flow Cytometry of Immune Cells

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Cells were harvested and washed before staining for extracellular markers including CD3, CD4, CD8, CD45RO, CD62L, CD127, and KLRG1 (BioLegend). For intracellular cytokine analysis, cells were stimulated with phorbol myristate acetate, ionomycin, and brefeldin A (Biolegend) for 4 hours. After stimulation, cells were stained with surface antibodies, followed by fixation with the BD fixation/permeabilization solution and staining with intracellular antibodies for IFNγ, TNF, IL‐4, IL‐10, and granzyme B (BioLegend) using 1X perm/wash solution according to the manufacturer's instructions. All samples were washed and analyzed using a FACS Canto II flow cytometer (BD). Data were analyzed using the FlowJo software (Version 10; TreeStar, Ashland).

Liu X., Yu Z., Wu Y., Shi S., Yao J., Feng X., Wen D., Shi Z., Zhao Z., Li Y., Zhou H., You C., Lin Y, & Yang M. (2021). The immune regulatory effects of tetrahedral framework nucleic acid on human T cells via the mitogen‐activated protein kinase pathway. Cell Proliferation, 54(8), e13084.

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Multiparametric Immune Profiling of T Cells

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Cells were stained with CD3 (BioLegend, clone: HIT3a), CD8 (BioLegend, clone: SK1), CD45RO (BioLegend, clone: UCHL1), T-bet (BioLegend, clone: 4B10), Eomes (R&D Systems, clone: 644730), NFATC1 (BioLegend, clone: 7A6), PD-1 (BioLegend, clone: EH12.2H7), TIM-3 (BioLegend, clone: F38-2E2), CTLA4 (BioLegend, clone: L3D10), LAG3 (BioLegend, clone: 11C3C65), Bcl-XL (Abcam, clone:7B2.5), FABP5 (R&D Systems, clone: 311215)，CPT1α (Proteintech, 15184-1-AP), CD137 (BioLegend, clone: 4B4-1), mito Tracker Red CMXROS (Invitrogen, M7512), TMRM (Invitrogen, M20036), LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, L3224), CD27 (BioLegend, clone: M-T271), CD127 (eBioscience, clone: eBioRDR5), IFNγ (BD Pharmingen, clone: B27), TCF7 (BioLegend, clone: 7F11A10), IRF4 (BioLegend, clone: IRF4.3E4), Blimp1 (BD Pharmingen, clone: 6D3), BCL2 (Cell Signaling Technology, clone: 124), Ki67 (Abcam, clone: B126.1), PPARγ (Abcam, clone: EPR18516), Annexin V (BD Pharmingen, 556547), CFSE (BD Pharmingen, 565082), KLRG1 (BioLegend, clone: 2F1/KLRG1), as well as BODIPY 493/503 (Invitrogen, D3922) and BODIPY FL C16 (Invitrogen, D3821). For intracellular staining, cells were given PMA/ionomycin (BioLegend, 423303) re-stimulation and then intracellular staining was performed as previously described.6 (link) FACS analysis was performed on a BD FACS Aria II flow cytometer and analyzed by FlowJo V.6 software.

Liu F., Liu W., Zhou S., Yang C., Tian M., Jia G., Wang H., Zhu B., Feng M., Lu Y., Qiao T., Wang X., Cao W., Wang X., Shi Y, & Wu D. (2020). Identification of FABP5 as an immunometabolic marker in human hepatocellular carcinoma. Journal for Immunotherapy of Cancer, 8(2), e000501.

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T Cell Phenotyping by Flow Cytometry

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T cells were phenotyped using CD3, CD4, CD8, CD56, CD45RA, CD45RO, CCR7, CD62L, TIM-3, PD-1, LAG-3, KLRG-1, CD25 surface antibodies (BioLegend, San Diego, California and BD Biosciences, Franklin Lakes, New Jersey, USA) as previously described.17 33 (link) The cells were acquired using the Gallios Flow Cytometer or BD FACSCanto II, and results were analyzed using Kaluza software (Beckman Coulter) or FlowJo analysis software (FlowJo, Ashland, Oregon, USA)

Sharma S., Woods M., Mehta N.U., Sauer T., Parikh K.S., Schmueck-Henneresse M., Zhang H., Mehta B., Brenner M.K., Heslop H.E, & Rooney C.M. (2023). Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy. Journal for Immunotherapy of Cancer, 11(4), e006267.

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Multiparametric Immune Profiling via FACS

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Cells were stained with CD4 (BioLegend, clone: RPA‐T4), CD3 (BioLegend, clone: HIT3a), CD8 (BioLegend, clone: SK1), PD‐1(Biolegend, clone:EH12.2H7), CD45RO (Biolegend, clone:UCHL1), CD45RA(BD Pharmingen, clone:HI100), T‐bet (BioLegend, clone: 4B10), Eomes (R&D, 644730), Blimp(BD, cline: 6D3), NFATC (Biolegend, clone: 7A6), PFR (Biolegend, clone: dG9), CD16 (BD Pharmingen,clone: B73.1), CD56 (BD Pharmingen, clone: MY31), CD25 (BD Pharmingen, clone: 2A3), CD127 (BD Pharmingen, clone: HIL‐7R‐M21), IFN‐γ (BD Pharmingen, clone: B27) conjugated to various fluorochromes. FACS analysis was conducted on a BD FACS Aria II flow cytometer, and FlowJo v10 was used for analysis.

Yang Y., Liu F., Liu W., Ma M., Gao J., Lu Y., Huang L., Li X., Shi Y., Wang X, & Wu D. (2020). Analysis of single‐cell RNAseq identifies transitional states of T cells associated with hepatocellular carcinoma. Clinical and Translational Medicine, 10(3), e133.

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