Pe anti mouse cd25

Manufactured by BioLegend

Sourced in United States

PE anti-mouse CD25 is a fluorochrome-conjugated antibody that binds to the CD25 antigen on the surface of mouse cells. CD25 is the alpha subunit of the interleukin-2 receptor and is expressed on activated T cells, regulatory T cells, and other immune cell types. This product can be used for the identification and analysis of CD25-positive cells in flow cytometry applications.

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CD25-PE (55 citations) Anti-CD25-PE (36 citations) Anti-mouse CD25 (9 citations) PE-conjugated anti-mouse CD25 (4 citations) PE/Cy7 anti-mouse CD25 antibody (2 citations) PE anti-mouse CD25 Antibody (2 citations) PE/Cy7-anti-mouse CD25 (2 citations) PE/CY5-anti-mouse CD25 (2 citations)

9 protocols using pe anti mouse cd25

Comprehensive Immunological Profiling of Tumor Cell Lines

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4T1, H22, HepG2, NIH‐3T3, and Panc02 were purchased from BeNa Culture Collection, China. Anti‐mouse PD‐1 antibody (PD‐1 Ab, BE0146‐100 mg) was purchased from Bio X Cell. Anti‐human PD‐1 Ab was obtained as a gift from Livzon Pharmaceutical Group Inc., China. Flow cytometry antibodies included PE anti‐mouse CD274 (B7‐H1, PD‐L1) (124308), APC/Cyanine7 anti‐mouse CD45 (103132), FITC anti‐mouse CD3 (100204), Brilliant Violet 421 anti‐mouse CD4 (100563), PE/Cy7 anti‐mouse CD8a (100722), PE anti‐mouse IFN‐γ (505808), Alexa Fluor 647 anti‐human/mouse Granzyme B (515406), APC anti‐mouse Ki‐67 (652406), PE anti‐mouse/human CD44 (103008), PE anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) (108407), APC/Cyanine7 anti‐mouse/human CD11b (101226), Brilliant Violet 421 anti‐mouse F4/80 (123137), PE anti‐mouse CD86 (105008), PE/Cy7 anti‐mouse CD206 (MMR) (141720), Alexa Fluor 647 anti‐mouse FOXP3 (126408), APC anti‐mouse CD49b (pan‐NK cells) (108910), PE anti‐mouse CD25 (102008), PE anti‐mouse/human CD44 (103008), APC anti‐mouse CD62L (104412), Zombie Aqua Fixable Viability Kit (423102), Cell Activation Cocktail (with Brefeldin A) (423304), True‐Nuclear Transcription Factor Buffer Set (424401), and Intracellular Staining Permeabilization Wash Buffer (10X) (421002) were purchased from BioLegend.

Liu X., Ye N., Liu S., Guan J., Deng Q., Zhang Z., Xiao C., Ding Z., Zhang B., Chen X., Li Z, & Yang X. (2021). Hyperbaric Oxygen Boosts PD‐1 Antibody Delivery and T Cell Infiltration for Augmented Immune Responses Against Solid Tumors. Advanced Science, 8(15), 2100233.

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Isolation and Activation of Murine Tregs

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Firstly, splenocytes were isolated from adult C57BL/6 and GFP C57BL/6 mice by pressing spleens through 70 μm filters (BD Bioscience, San Jose, CA, USA). Secondly, CD4⁺ T cells were sorted using Mouse CD4⁺ T cell Pre-enriched Kit (Stemcell, Vancouver, BC, Canada) via magnetic activated cell sorting (MACS). CD4⁺/CD25⁺ Tregs were then enriched from CD4⁺ T cells by Flow Activated Cell Sorter (FACS; BD Bioscience) after being labeled with PE anti-mouse CD25 (Biolegend, San Diego, CA, USA). Enrichment of CD4⁺/CD25⁺ Tregs was further confirmed by Foxp3 staining (Biolegend). These cells were then cultured in the presence of anti-mouse CD28 (2 μg/ml, Ebioscience) and recombinant mouse IL-2 (400 U/ml, Life Technologies, Grand Island, NY, USA) in cell culture plates pre-coated with anti-mouse CD3e (10 μg/ml, Ebioscience, Franklin Lakes, NJ, USA). Three days later, CD44 and CD62L (Biolegend) staining were performed to confirm Treg activation. At the same time, mRNA level of IL-10, TGF-β and epstein-barr virus induced gene-3 (ebi3) were evaluated by qRT-PCR.

Wang J., Xie L., Yang C., Ren C., Zhou K., Wang B., Zhang Z., Wang Y., Jin K, & Yang G.Y. (2015). Activated regulatory T cell regulates neural stem cell proliferation in the subventricular zone of normal and ischemic mouse brain through interleukin 10. Frontiers in Cellular Neuroscience, 9, 361.

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Flow Cytometry Analysis of Transduced T Cells

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Flow cytometry was performed to evaluate transduction of T cells by our retroviral and lentiviral vectors. F8CAR expression was characterized by first incubating cells with human FVIII protein. Cells were then stained with FITC conjugated anti-human FVIII antibody (Affinity Biologicals, ON, Canada). Transduced cells were stained with Alexa Fluor 700 anti-mouse CD4 (Thermo Fisher), PE anti-mouse CD25 (BioLegend San Diego, CA), Brilliant Violet 510™ anti-mouse CD45.1 (BioLegend), PE-Cyanine7 anti-mouse CD45.2 (Thermo Fisher), and Alexa Fluor 647 anti-mouse Foxp3 (Thermo Fisher) antibodies. eFluor 450 fixable viability dye (eBioscience San Diego, CA) was used to gate out dead cells. Stained cells were analyzed on an LSRII Flow Cytometer (BD Biosciences) and data compiled on FlowJo software (TreeStar). Stained naive mouse cells were used as compensation controls and also normalization controls at different time points. Cell sorting was performed on the FACSAria (BD Biosciences).

Fu R.Y., Chen A.C., Lyle M.J., Chen C.Y., Liu C.L, & Miao C.H. (2020). CD4+ T Cells Engineered with FVIII-CAR and Murine Foxp3 Suppress Anti-Factor VIII Immune Responses in Hemophilia A Mice. Cellular immunology, 358, 104216.

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Phenotypic Analysis of Immune Cells in NOD Mice

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Spleen cells and pancreatic lymphocytes were isolated from spleen and pancreatic lymph node (PLN) of 39-week-old NOD mice by mechanical disruption. Cells were respectively stained for Treg [(CD4⁺CD25⁺Foxp3⁺) (Foxp3 is a necessary factor for the development and function of Treg cells, and it is also the most sensitive marker for Treg cells)], Th17 (CD4⁺ Rorγt⁺), Th1 (CD4⁺ T-bet⁺) or Th2 (CD4⁺ GATA3⁺) phenotypes. The following antibodies were used: FITC anti-mouse CD4 (BioLegend Inc. CA, USA), PE anti-mouse CD25 (BioLegend Inc. CA, USA), APC anti-GATA3 (BioLegend Inc. CA, USA), Alexa Fluor 647 Mouse Anti-Mouse RORγt (BD Biosciences, CA, USA), Alexa Fluor 647 Mouse anti-T-Bet (BD Biosciences, CA, USA), Alexa Fluor 647 Rat anti-Mouse Foxp3 (BD Biosciences, CA, USA). Stained cells were detected by Flow cytometer (Accuri C6, BD Biosciences, CA, USA) and analyzed using BD Accuri C6 systerm Software.

Gao H., Zhao Q., Tang S., Li K., Qin F., Song Z., Pan Y., Jin L, & Zhang Y. (2021). Continuous stimulation of dual-function peptide PGLP-1-VP inhibits the morbidity and mortality of NOD mice through anti-inflammation and immunoregulation. Scientific Reports, 11, 3593.

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Multicolor Immunofluorescence Tumor Analysis

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Tumors were collected and fixed with 4% paraformaldehyde and cut into 5 μm thick sections. FITC anti-mouse CD8a antibody, PE anti-mouse CD25, and FITC anti-mouse CD206 (MMR) antibody (BioLegend) were used to stain samples. Then, they were analyzed by fluorescence microscopy.

Wu J., Wang S., Liu S., Liu F, & Zhou F. (2022). Immunoadjuvant Nanoparticles as Trojan Horses for Enhanced Photo-Immunotherapy in the Treatment of Triple-Negative Breast Cancer. Frontiers in Pharmacology, 13, 883428.

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Targeted Delivery of Abemaciclib and IMD-0354

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Abemaciclib was purchased from MedChemExpress Co. Ltd. IMD‐0354 was purchased from Selleckchem Co. Ltd. MAL‐PEG₂₀₀₀‐NHS was purchased from Beijing Kaizheng Biotech Co. Ltd (Beijing, China). Poly (L‐lysine) (PLL, M_w = 3–7 w), 2,3‐Dimethylmaleic Anhydride (DMA), Cis‐aconitic acid anhydride (CA), 3,4,5,6‐Tetrahydrophthalic anhydride (TDA) and succinic anhydride (SA) were purchased from Sigma‐Aldrich Co. Ltd (American). NGR peptide (sequence: GCNGRCGC) was obtained from Shanghai Apeptide Co. Ltd (Shanghai, China). Polyamindoamine (PAMAM‐G5, M_w = 28 826) was purchased from CY dendrimer technology Co. Ltd (Weihai, China). Cell Cycle and Apoptosis Analysis Kit was the product of Beyotime Biotechnology Co. Ltd (Shanghai, China). Alexa Fluor 647 anti‐mouse F4/80, Brilliant Violet 421 anti‐mouse CD206, PerCP/Cy5.5 anti‐mouse F4/80, Alexa Fluor 488 anti‐mouse CD86, APC anti‐mouse CD206, APC anti‐mouse CD3, FITC anti‐mouse CD4, PE anti‐mouse CD8, PE anti‐mouse CD25, and Alexa Fluor 488 anti‐mouse Foxp3 were purchased from Biolegend. ELISA kits were obtained from Dakewei Co. Ltd (Nanjing, China). All other reagents and solvents were obtained from Sinopharm Co., Ltd (Shanghai, China) and Sigma‐Aldrich Co., Ltd (Shanghai, China).

Yang R., Zhang Z., Fu S., Hou T., Mu W., Liang S., Gao T., Guan L., Fang Y., Liu Y, & Zhang N. (2020). Charge and Size Dual Switchable Nanocage for Novel Triple‐Interlocked Combination Therapy Pattern. Advanced Science, 7(18), 2000906.

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Flow Cytometric Isolation and Analysis of Murine Regulatory T-cells

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Total mesenteric lymph node cells of male C57BL/6 mice were isolated by pushing through a cell strainer. After fixation, permeabilization and washing, the cells were stained with anti-mouse CD4 PerCP/Cy5.5 (Biolegend 116012), anti-mouse CD45 BV650 (Biolegend 103151), anti-mouse TCR β chain PE/Cy7 (Biolegend 109222), anti-mouse CD8α BV510 (Biolegend 100752), anti-mouse Foxp3 BV421 (Biolegend 126419), the fixable viability dye eFluor780 (eBioscience 65-0865-14) and flow cytometrically measured using a LSR II Fortessa instrument followed by a detailed analysis using FlowJo software. For FACS-sorting of regulatory T-cells, mesenteric lymph node cells were stained with anti-mouse CD45 BV650 (Biolegend 103151), anti-mouse TCR β chain PE/Cy7 (Biolegend 109222), anti-mouse CD4 BV711 (Biolegend 100550), anti-mouse CD25 PE (Biolegend 101903), fixable viability dye eFluor780 (eBioscience 65-0865-14) and sorted on a FACS Aria. RNA of sorted cells was isolated using the RNeasy Mini Kit (Qiagen 74106), converted into cDNA, and subjected to TaqMan Real-Time PCR assay using the primers Mm03024075 (Hprt), Mm00475162 (Foxp3), Mm01178820 (Tgfb1) and Mm01288386 (IL10; all from Thermo Fisher Scientific). Samples were run on a Light Cycler 480 and normalized to the house-keeping gene Hprt.

Kyburz A., Urban S., Altobelli A., Floess S., Huehn J., Cover T.L, & Müller A. (2017). Helicobacter pylori and its secreted immunomodulator VacA protect against anaphylaxis in experimental models of food allergy. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 47(10), 1331-1341.

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Multiparameter Flow Cytometry Analysis

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Primary antibodies used for this study includes Annexin V-FITC (Biolegend Cat# 640906), anti-PD-1-PE (BioLegend Cat# 621607), anti-human CD8-PE (BioLegend Cat# 344706), anti-mouse CD4-PE (BioLegend Cat# 100408), anti-mouse CD25-PE (BioLegend Cat# 102012), anti-mouse FOXP3-Alexa Fluor® 488 (BioLegend Cat# 126405), anti-mouse CD8-APC (BioLegend Cat# 100711), anti-mouse PD-1-PE (BioLegend Cat# 135205), IgG isotype control-PE (BioLegend Cat# 400907), anti-human CD4-APC (eBioscience Cat# RPA-T4). FoxP3 staining used FoxP3 buffer (BioLegend Cat#421403). Samples were analyzed using FACSCalibur and FlowJo software.

Wu G., Deng W., Chen H.Y., Cho H.J, & Kim J. (2024). Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1. Scientific Reports, 14, 6625.

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Multicolor Flow Cytometry Panel

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Antibodies used include: anti-mouse CD45 APCCY7 (Clone 30-F11 Biolegend Cat #103116) and anti-mouse CD45 Eflour 450 (Clone 30-F11 Invitrogen Cat#48-0451-82); anti-mouse LY6G (Clone 1A8; Biolegend Cat#127623); anti-Mouse CD3 Pacific Blue (Clone 145-2C11 Biolegend Cat#100334); anti-Mouse CD3e, PerCP-Cy5.5 (Clone: 145-2C11, eBioscience, Cat#: 45-0031-82); anti-Mouse CD8a PerCP (Clone Ly-2 BDBiosciences Cat#M037858); anti-Mouse CD4, FITC (Clone: GK1.5; eBioscience, Cat#: 25-0041-82); anti-mouse CD19 PE (Clone eBio1D3 ThermoFisher Cat# 12-0193-83); anti-mouse CD11c APC (Clone N418; Biolegen Cat #117310); anti-mouse CD11b FITC (Clone M1/70.15 Invitrogen Cat #RM2801); anti-mouse CD25 PE (Clone 3C7 Biolegend Cat#101904); anti-mouse CD44 PE (Clone 1M7; Biolegend Cat# 10,008); anti-mouse CD62L APC (Clone Mel-14 Biolegend Cat# 104411).

Scieszka D.P., Garland D., Hunter R., Herbert G., Lucas S., Jin Y., Gu H., Campen M.J, & Cannon J.L. (2023). Multi-omic assessment shows dysregulation of pulmonary and systemic immunity to e-cigarette exposure. Respiratory Research, 24, 138.

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