Cd25 apc

Manufactured by BioLegend

Sourced in United States

CD25-APC is a fluorescently-labeled antibody that binds to the CD25 antigen, also known as the interleukin-2 receptor alpha chain (IL-2Rα). CD25 is expressed on activated T cells, B cells, and natural killer cells. The APC (Allophycocyanin) fluorescent dye allows for the detection and analysis of CD25-positive cells using flow cytometry or other immunoassay techniques.

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57 protocols using cd25 apc

Activation and Co-culture of T Cells with Myeloma

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T cells were isolated from healthy donor using CD3 magnetic beads (Miltenyi Biotec). CD3 T cells (10⁶ cells) were activated with 30μl of CD3/CD28 beads (Invitrogen) in T cell medium (X-VIVO medium +5% FBS+1% penicillin/streptomycin supplemented with human IL2 400U/ml (Peprotech) for 3-4 days. Magnetic beads were removed, and T cells were stained with cell trace (CFSE, Invitrogen), following manufacturer’s manual. Irradiated myeloma cells were plated into the T cell culture at a ratio of 1 myeloma: 2 T cells. For non-contact co-culture of T cells and MM cells, MM cells were cultured in the upper chamber of transwell insert (pore size, 3 μM, #09-761-80, Thermo Fisher) in U-bottom 96 well plate. 4 days activated T cells were cultured in the lower chambers of 96-well transwell plate. 48 hours later, cells were stained with CD3-FITC, CD25-APC, and CD69-PE (Biolegend), and analyzed by flow cytometry (Atune NxT, Invitrogen).

Nguyen H.P., Le A.Q., Liu E., Cesarano A., DiMeo F., Perna F., Kapur R., Walker B.A, & Tran N.T. (2023). Protein arginine methyltransferase 1 is a therapeutic vulnerability in multiple myeloma. Frontiers in Immunology, 14, 1239614.

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Comprehensive Tumor Immune Profiling

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4T1 tumors were excised and digested with the Mouse Tumor Dissociation kit (Miltenyi Biotec) as per manufacturer’s instructions, and ran on a Miltenyi gentleMACS Octo Dissociator with Heaters using pre-set program (37C_m_TDK2). The resulting cell suspensions were filtered using a 40 µm cell strainer and subjected to RBC lysis. Samples were counted and stained with the Zombie Aqua Fixable Viability Dye (BioLegend) to distinguish live cells. All samples were then incubated with purified anti-mouse CD16/32 (Fc block) prior to staining. The following anti-mouse antibodies, all purchased from BioLegend, were used for immunostaining in the indicated dilutions: CD69 APC (Clone H1.2 F3) 1:100, CD4 PE/Cy5 (Clone GK1.5) 1:100, FOXP3 Alexa Fluor 488 (Clone 150D) 1:50, CD25 APC (Clone PC61) 1:100, CD45 APC-Cy7 Clone 30-F11 (1:500), CD3 BV421 Clone 145–2 C11(1:100), CD8 FITC Clone 53–6.7 (1:100), CD11b PerCP-Cy5.5 Clone M1/70 (1:200), Ly6G Alexa Fluor 488 (Clone 1A8) 1:100, Ly6C Brilliant Violet 421 (Clone HK1.4) 1:100, CD4 PerCP-Cy5.5 Clone GK1.5 (1:100). Flow data were acquired using a MACSQuant Analyzer 10 and analyzed using FlowJo version 10.1 (Tree Star).

Pilones K.A., Hensler M., Daviaud C., Kraynak J., Fucikova J., Galluzzi L., Demaria S, & Formenti S.C. (2020). Converging focal radiation and immunotherapy in a preclinical model of triple negative breast cancer: contribution of VISTA blockade. Oncoimmunology, 9(1), 1830524.

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Multiparametric Flow Cytometry Immunophenotyping

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Mouse FITC-CD4 and eFluor 605-CD44 [BD-Pharmingen]; KJ1.26 anti-TCR (Caltag Laboratories), Brilliant Violet 570-CD4 and Alexa Fluor 700-CD44 were from [Biolegend], FITC-Klotho,PE-Cy7-CD99, PE-Cy5-VDR, and PE-Cy7-CNR2 were from [Bioss]; APC-CCR10 and APC-ITGA3 were from [R&D Systems]; Fixable viability dye eFluor 780 was from [eBioscience]. Antibodies for human PBMC staining were as follows: from Biolegend, CD99-FITC,CD69-BV421 and CD69-APC , CD45R0-APC and CD45R0-BV421, CD3-AF700, CD4-Pe/Cy7, CD45RA-PE, Itga3 (CD49c)-PE, IL-7R-BV510, CD25-APC, HLA-DR-APC. From BD Biosciences, CCR10-APC and CCR10-PerCP Cy5.5. and viability dye eFluor 780 as above. Relevant mouse anti human isotype antibodies to IgG1k or IgG2ak were utilized for the following fluorophores: BV421, BV510, FITC, PE, and APC for both isotypes (Biolegend).

Song N., Sengupta S., Khoruzhenko S., Welsh R.A., Kim A., Kumar M.R., Sønder S.U., Sidhom J.W., Zhang H., Jie C., Siliciano R.F, & Sadegh-Nasseri S. (2020). Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence. Cellular immunology, 357, 104210.

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Isolation of Mouse Colonic Immune Cells

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Surgically removed fresh 1-cm mouse colon tissues were opened and washed with cold PBS to remove the fecal contents. The tissues were quickly transported to the centrifuge tube containing 10 mL 1640 medium (10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S), 1 mM EDTA) on an orbital shaker at 300 rpm for 30 min at 37°C. After washing, the colons were finely minced and digested with 15 mL of HBSS containing 10% FBS, 1.5 mg/mL Type-VIII Collagenase (C2139; Millipore, Sigma), and 40 μg/mL DNase I at 300 rpm for 15 min at 37°C. After the digestion, the digested colonic lamina propria cells were filtered through a 100-μm strainer, centrifuged at 1500 rpm for 5 min at 4°C, and resuspended in 2 mL PBS for flow cytometric analysis.^{82 (link)}The antibodies as follows: (1) CD45-Alexa Fluor 700 (103,128, Biolegend), MHCII-APC (107,613, Biolegend), CD11c-PE (117,307, Biolegend), F4/80-FITC (123,108, Biolegend), CD11b-Percp-Cy5.5 (101,227, Biolegend); (2) CD45-Alexa Fluor 700, CD3-FITC (100,203, Biolegend), CD25-APC (101,909, Biolegend), NK1.1-PE (108,707, Biolegend).

Liu C., Huang S., Wu Z., Li T., Li N., Zhang B., Han D., Wang S., Zhao J, & Wang J. (2021). Cohousing-mediated microbiota transfer from milk bioactive components-dosed mice ameliorate colitis by remodeling colonic mucus barrier and lamina propria macrophages. Gut Microbes, 13(1), 1903826.

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Phenotypic Analysis of Murine Regulatory T Cells

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Half a million splenocytes were stained in each FACS tube. First, cells were washed with staining buffer (Biolegend, San Diego, CA, USA), and centrifuged at 800 g for 5 min at 4 °C. They were then stained with fluorochrome-conjugated antibodies after blocking of Fc receptors (CD16/32, Biolegend, San Diego, CA, USA) for 5 minutes. The following antibodies were used: FoxP3-PE, CD3-PE/Cy7, CD25-APC, CD4-PB, CD8a-FITC and CD3-PE-Cy7, all from Biolegend (San Diego, CA, USA). The cells were stained 30 min on ice in the dark with antibodies binding extracellular targets. Cells were then washed to remove unbound antibodies, incubated with Fixation/Permeabilization buffers (eBioscience, San Diego, CA, USA) for 30 min on ice and blocked with CD16/32 prior incubation with anti-FoxP3 for 30 min. Cells were resuspended in PBS buffer containing 1% BSA and 0.5 mM EDTA. Cells were run on Gallios flow cytometer (Beckman Coulter, High Wycombe, UK) and analyzed with FlowJo software V.10 (Tree Star, Inc. Ashland, OR, USA).

Vallejo J., Dunér P., To F., Engelbertsen D., Gonçalves I., Nilsson J, & Bengtsson E. (2019). Activation of immune responses against the basement membrane component collagen type IV does not affect the development of atherosclerosis in ApoE-deficient mice. Scientific Reports, 9, 5964.

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Comprehensive Immune Cell Profiling

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We used the following antibodies: CD4-PErCP (100538), CD8-APC/Cy7 (100713), CD69-FITC (104506), CD25-APC (101910), IFNγ-APC (505810), IL17-PE/Cy7 (506922), CD11b-APC/Cy7 (101226), CD11c-APC (117310), CD80-FITC (104706), MHCII-PE (107607), CD3-PacificBlue (100214), CD4-PE (100512), CD4-APC (100516) and CD4-FITC (100510) from Biolegend, USA.
SIRT2 (ab211033), β-actin (ab8227), acH3K18 (ab40888), α-tubulin (ab184613), acetyl-α-tubulin (ab179484), acetyl-NFκB p65 (ab19870), Alexa Fluor 647 (ab150075) and IgG-APC isotype control (ab232814) from Abcam.
ERK1/2 (9102), Phospho-ERK1/2 (9101), p-38 (9212) and phospho-p38 (4511) from Cell Signaling Technology.

Bhaskar A., Kumar S., Khan M.Z., Singh A., Dwivedi V.P, & Nandicoori V.K. (2020). Host sirtuin 2 as an immunotherapeutic target against tuberculosis. eLife, 9, e55415.

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Multicolor Flow Cytometry of Immune Cells

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Bone marrow cells (0.5 × 10⁶) were stained with B220-FITC (Becton Dickinson (BD) Pharmingen, Franklin Lakes, NJ, USA), IgM-PE (Southern Biotechnology Associates, Inc., Birmingham, AL, USA), c-kit (CD117)-APC (Biolegend, San Diego, CA, USA), CD25-APC (Biolegend), CD19-Brilliant Violet 421 (Biolegend), and CD93-PE-Cy7 (Biolegend). Splenocytes (0.5 × 10⁶) were stained with B220-FITC (BD Pharmingen), CD21-FITC (BD Bioscience), IgM-PE (Southern Biotechnology Associates, Inc.), CD93-APC (eBioscience, Vienna, Austria), CD19-Brilliant Violet 421 (Biolegend), and CD23-PE-Cy7 (eBioscience). All cells were analyzed in a FACS Canto II (BD) and data were further processed in Flow Jo version 10.0.4 (Three Star, Inc., Ashland, USA). All analyses started with a singlet gate, thereafter a lymphocyte gate and gates for indicated populations. Results are presented as absolute number of cells of the different populations.

Bernardi A.I., Andersson A., Grahnemo L., Nurkkala-Karlsson M., Ohlsson C., Carlsten H, & Islander U. (2014). Effects of lasofoxifene and bazedoxifene on B cell development and function. Immunity, Inflammation and Disease, 2(4), 214-225.

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Immunophenotyping of GMSCs and Splenocytes

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GMSCs were collected and suspended in cell staining buffer (0.5% BSA in PBS with 2 mM EDTA) followed by incubation with CD14 (PE), CD19 (PerCP-Cy5.5), CD29 (APC), CD34 (PE), CD44 (FITC), CD45 (PE), CD73 (PE), CD90 (FITC), and CD105 (PE) antibodies (Biolegend) in the dark at room temperature for 30 min. For intracellular staining, splenocytes from mice were first stained with CD3 (APC/Cy7), CD4 (FITC), and CD25 (APC) antibodies (Biolegend) and then fixed, permeabilized, and stained intracellularly for IL-4 (PE/Cy7) and IL-17 (PE). After staining, cells were washed twice with PBS and submitted to flow cytometric analysis (BD). Data were analyzed using the FlowJo 7.6 software.

Ye Z., Liang Y., Lin B., Li Y., Chai X., Lian J., Zhang X., Che Z, & Zeng J. (2022). Gingiva-Derived Mesenchymal Stem Cells Attenuate Imiquimod- (IMQ-) Induced Murine Psoriasis-Like Skin Inflammation. Stem Cells International, 2022, 6544514.

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Isolation and Phenotyping of Mouse Orbital Lymph Nodes

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Draining lymph nodes of mouse orbits were harvested and teased apart into a single-cell suspension by mashing tissue between two frosted microscope slides. Collected cells were filtered using a 100-μm cell strainer to eliminate clumps and before centrifuging 5 min 400×g at 4 °C. Cell pellet was then resuspended in 1 ml of PBS with 2% FBS to count and evaluate cell viability. Appropriate volumes of each sample equal to 2 × 10⁶ cells were transferred to new tubes for staining. Antibody mix of CD4 FITC, CD8PE and CD25 APC (all from BioLegend) was added to each sample and added up to 100 μl using PBS with 2% FBS. Cells were incubated for at least 30 min at 2–8 °C before washing and spinning down at 400×g for 5 min. Cells were resuspended in 300 μL of PBS with 2% FBS and subjected to read by flow cytometry.

Moshkelgosha S., Verhasselt H.L., Masetti G., Covelli D., Biscarini F., Horstmann M., Daser A., Westendorf A.M., Jesenek C., Philipp S., Diaz-Cano S., Banga J.P., Michael D., Plummer S., Marchesi J.R., Eckstein A., Ludgate M, & Berchner-Pfannschmidt U. (2021). Modulating gut microbiota in a mouse model of Graves’ orbitopathy and its impact on induced disease. Microbiome, 9, 45.

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Comprehensive Treg Phenotyping by Flow Cytometry

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Antibodies used for flow cytometry were as follows: CD4-Pacific Blue, CD25-PE, CD25-APC, CD127-FITC, Foxp3-PE, CD38-PE-Cy7, AnnexinV-PE, PD1-APC, CD8-FITC, CTLA4-PE-Cy7, CD44-FITC, CD62L-FITC, ICOS-FITC, GITR-PE, OX40-FITC, CD138-FITC, PD-L1-PE, and their isotype-matched mAbs (all from Biolegend). Intracellular staining of Foxp3, CTLA4, GITR, and OX40 were performed after fixation and permeabilization using cytofix/cytoperm kit (BD Biosciences), according to manufacturer’s protocol. Tregs were gated as CD4+CD25highFoxp3+ cells in CD4+ population and then sequential markers were assayed on Tregs, whereas CD4+CD25− cells were identified as Tcons. The remaining CD4+CD25low/intermediate subset was excluded in the current study because of their limited immunosuppressive activity compared with CD25high population (34 (link)). To avoid the effect of permeabilization when apoptosis assay was performed, CD4+CD25highCD127low/− cells were identified as Tregs (35 (link)). All flow cytometry was performed by BD FACS CantoII, and analyzed on FlowJo software version 10 (Treestar).

Feng X., Zhang L., Acharya C., An G., Wen K., Qiu L., Munshi N., Tai Y.T, & Anderson K.C. (2017). Targeting CD38 suppresses induction and function of T regulatory cells to mitigate immunosuppression in multiple myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(15), 4290-4300.

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