Il 2 pecy7

Manufactured by BD

Sourced in United States

IL-2-PECy7 is a fluorescently-labeled antibody specific for the cytokine interleukin-2 (IL-2). It can be used to detect and quantify IL-2-expressing cells in flow cytometry applications.

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Anti-IL-2-PEcy7 (2 citations)

5 protocols using il 2 pecy7

Multiparametric Flow Cytometry Analysis

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The fluorochrome labeled monoclonal antibodies (mAbs) anti-CD2-FITC, anti-CD3-Alexa 700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-APC Fluor 780, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20 PECy7, anti-CD19-V450, anti-CD20-APCCy7, anti-CD25-PEcy7, anti- CD28-PE, anti-CD28-FITC, anti-CD38-PE, anti-CD39-FITC, anti-CD45-PerCP, anti-CD56-APC, anti-CD57-FITC, anti-CD197-PECy7, anti-HLA-DR, anti-IgM, anti-IgD, anti-Ki67-FITC, anti-Bcl-2-PE, anti-TNF-α-PE, anti-TNF-α-APC, IFN-γ-PcpCy5.5, and IL-2-PECy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-CD45RA-QDOT655 and anti-CD69-FITC mAb were obtained from Invitrogen (Carlsbad, CA). Anti-CD8-Alexa780, anti-CD27-Alexa Fluor 700, and anti-CD38-eFluor 650NC were obtained from E-bioscience (San Diego, CA). Anti-CD24-PEcy7 and anti-CD279-PE (PD-1) were purchased from Biolegend (San Diego, CA).
Human EBV protein and CMV peptide pool of pp65 sequence consisting of 138 peptides (15 mers with 11 amino acid overlaps) was purchased from JPT Peptide Technologies (Berlin, Germany).
All patients were DSA-free with a calculated panel reactive antibody (PRA) ≤20% at enrollment. Patient samples were assessed for donor-specific alloantibody post-transplantation as described previously ^{(18 (link))}.

Xu H., Samy K.P., Guasch A., Mead S.I., Ghali A., Mehta A., Stempora L, & Kirk A.D. (2015). Post Depletional Lymphocyte Reconstitution During Belatacept and Rapamycin Treatment in Kidney Transplant Recipients. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(2), 550-564.

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Cytokine Response Profiling in Immunized Mice

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Spleen, draining lymph nodes and lungs were isolated from immunized mice 7 days after the last immunization. To identify cytokine-producing cells, single cell suspension was prepared from each organ as previously described (35 (link)) and re-stimulated with 5 μg/ml of OMPOT for 6 h. To accumulate cytokines, Golgi plug (BD Biosciences, San Joes, CA, USA) was added and incubated for additional 10 h. The cells were stained with anti-mouse CD4-PerCP and CD44-FITC and stained with anti-mouse IFN-γ-APC, IL-17-PE, TNF-α-Pe-Cy7, or IL-2-Pe-Cy7 (BD Biosciences) after the fixation and permeabilized using Cytofix/Cytoperm solution. The cells were acquired by using flow cytometry, LSR II (BD Biosciences). Cytometry data were analyzed by using FlowJo software (Tree Star, Ashland, OR, USA). Comparison of cytokines combination and Pie graphs were analyzed using Simplified Presentation of Incredibly Complex Evaluations (Spice) software version 5.3 (National Institutes of Health, Bethesda, MD, USA).

Park S.M., Gu M.J., Ju Y.J., Cheon I.S., Hwang K.J., Gill B., Shim B.S., Jeong H.J., Son Y.M., Choi S., Jeung W., Han S.H., Chu H, & Yun C.H. (2020). Intranasal Vaccination with Outer-Membrane Protein of Orientia tsutsugamushi induces Protective Immunity Against Scrub Typhus. Immune Network, 21(2), e14.

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Cytokine Profiling of Activated PBMCs

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PBMC (1×10⁶ cells) were cultured in complete culture medium [RPMI (Corning) supplemented with 10% fetal bovine serum, 100mg/ml streptomycin and 100U/ml penicillin] and were stimulated with phorbol 12-myristate 13-acetate (PMA 25 ng/ml) and ionomycin (0.5 µg/ml) for 6 hours in the presence of Brefeldin A (GolgiPlug, BD). At the end of stimulation, cells were washed, stained for dead cells using the Zombie Aqua Fixable Viability Kit (20 min, 4°C) (Biolegend) and then labeled with the following antibodies: CD3 Brilliant Violet Horizon 395, CD4 PerCP eFluor 710, CD8 PerCP, CD45RA APC Cy7 and CCR7 Alexa Fluor 488 (30 min, 4°C). Subsequently, cells were permeabilized (20 min, 4°C)(Cytofix/Cytoperm, BD), washed twice (Perm/Wash; BD) and stained with IL-2 PE Cy7, IFNγ Pacific Blue, IL-17A Alexa Fluor 647 and IL-4 PE (30 min, 4°C). Antibodies were purchased from BD Bioscience, Biolegend and eBioscience.

Comte D., Karampetsou M.P., Kis-Toth K., Yoshida N., Bradley S.J., Kyttaris V.C, & Tsokos G.C. (2017). CD4+ T cells from SLE patients respond poorly to exogenous IL-2. Arthritis & rheumatology (Hoboken, N.J.), 69(4), 808-813.

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Profiling Cytokine Responses in Activated PBMCs

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PBMCs were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum and stimulated with anti-CD3/CD28 (2 and 2.5 μg/mL) at the presence of Plus Golgiplug (BD Pharmingen, San Diego, CA, USA) for 5 hours. Cell viability was assessed using the Fixable viability dye eFluor^TM 506 (invitrogen, Carlsbad, CA, USA). Cells were then surface stained with CD4-FITC, CD8-APC-H7, PD-1-BV785, and CD26-PE-CF594. After fixation and permeabilization, intracellular staining was performed with IL-2-PE-Cy7, TNF-α-BV421, IFN-γ-APC (BD Biosciences) antibodies.

Zhou H., Jia B., Annageldiyev C., Minagawa K., Zhao C., Mineishi S., Ehmann W.C., Naik S.G., Cioccio J., Wirk B., Songdej N., Rakszawski K.L., Nickolich M.S., Shen J, & Zheng H. (2023). CD26lowPD-1+ CD8 T cells are terminally exhausted and associated with leukemia progression in acute myeloid leukemia. Frontiers in Immunology, 14, 1169144.

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T Cell Cytokine Induction Assay

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Cryopreserved PBMC were thawed, washed, resuspended in RPMI + 10% FBS (R10), and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and incubated for 2 hrs with our panel of 10 LRA. Following this 2-hour incubation period, GolgiPlug (BD, cat. no. 555029) and GolgiStop (BD, cat. no. 554724) were added to each condition at a concentration of 1:1000 and 6:10000 respectively, then incubated at 37°C for an additional 16 hrs. Cells were harvested, pelleted, resuspended, stained for 20 minutes at 4°C with surface staining mAb (CD3-BV605, CD4-BV421, CD8-PerCp-Cy5.5 and LIVE/DEAD Near-IR stain), fixed and permeabilized as described above, and then stained for 30 minutes at 4°C with the intracellular mAb TNF-α- PE/Dazzle-594 (Biolegend cat. no. 502946), IL-2- PE/Cy7 (BD cat. no. 560707) and IFN-γ- BV510 (Biolegend cat. no. 502544). PBMC were washed and fixed in (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis, as described above.

Lilie T., Bouzy J., Asundi A., Taylor J., Roche S., Olson A., Coxen K., Corry H., Jordan H., Clayton K., Lin N, & Tsibris A. (2023). Opioid use does not limit potent low-dose HIV-1 latency reversal agent boosting. medRxiv.

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