Il 2 percp cy5

Manufactured by BioLegend

IL-2-PerCP-Cy5.5 is a fluorescently-labeled antibody for the detection of interleukin-2 (IL-2) in flow cytometry applications. It is conjugated with the PerCP-Cy5.5 fluorochrome.

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Lab products found in correlation

Cd3 apc h7, by BD (3 mentions) Cd4 pe cy5, by BD (2 mentions) Cd8 bv785, by BioLegend (2 mentions) Il 22 apc, by Thermo Fisher Scientific (2 mentions) Ifn γ alexa fluor 700, by Thermo Fisher Scientific (2 mentions) Live dead fixable aqua blue, by Thermo Fisher Scientific (2 mentions) Il 17 alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Mip 1β pe cy7, by BD (2 mentions) Transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Cd25 pe cy5, by BioLegend (1 mentions) Tnf α pe cy7, by BD (1 mentions) Il 4 pe, by BD (1 mentions) Il 2 pe, by BD (1 mentions) Cd8 pb, by BD (1 mentions) Cd8 efluor625nc, by Thermo Fisher Scientific (1 mentions) Il 13 pe, by BioLegend (1 mentions) Il 5 pe, by BioLegend (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Cd4 fitc, by BioLegend (1 mentions) Cd3 apc, by BioLegend (1 mentions)

Close spelling variants of this product

PerCP-Cy5.5 (133 citations)

5 protocols using il 2 percp cy5

Comprehensive Immunophenotyping of Antigen-Specific CD4 T Cells

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6-day stimulated, CFSE-labeled PBMCs were subjected to three different panels of staining to measure: phenotypic and cytokine profile, HIV infectivity (p24) and intracellular transcription factors (T-bet and EOMES) in CFSE-low, Ag-specific CD4 T cells. Briefly, cells were first stained for viability with LIVE/DEAD Fixable Aqua Blue (Life Technologies), followed by surface makers staining, including CD3-APC-H7 (BD Bioscience), CD4-PE-Cy5 (BD Bioscience), CD8-BV785 (Biolegend), CD25-PE-Cy5 (Biolegend), CCR5-Pacific Blue (BD Bioscience), α4β7-APC (NIH AIDS Reagent Program), CCR6-APC (R&D Systems), CCR9-PerCP-Cy5.5 (Biolegend). After surface staining, cells were fixed, permeabilized (BD Bioscience) and subjected to intracellular staining, including HIV p24-PE (Beckman Coulter), IL-17-Alexa Fluor 488 (eBioscience), IL-22-APC (eBioscience), IL-2-PerCP-Cy5.5 (Biolegend), IFN-γ-Alexa Fluor 700 (eBioscience), MIP-1β-PE-Cy7 (BD Bioscience). Combination of surface and intracellular staining antibodies may vary according to different assays. For intracellular staining for T-bet and EOMES, cells were fixed and permeabilized using eBioscience’s transcription factor staining buffer set (eBioscience) according to manufacturer’s instructions. Cells were then subjected to intracellular staining with anti-T-bet-Pacific Blue (Biolegend) and anti-EOMES PE-eFluor 610 (eBioscience).

Liu F., Fan X., Auclair S., Ferguson M., Sun J., Soong L., Hou W., Redfield R.R., Birx D.L., Ratto-Kim S., Robb M.L., Kim J.H., Michael N.L, & Hu H. (2016). Sequential Dysfunction and Progressive Depletion of Candida albicans-Specific CD4 T Cell Response in HIV-1 Infection. PLoS Pathogens, 12(6), e1005663.

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Multiparameter Flow Cytometry Panel

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The following monoclonal antibodies (mAbs) were used in different combinations. CD3-APC-H7 (CloneSK7), CD4-PECF594 or CD4-APC (Clone RPA-T4), CD8-PB (Clone RPA-T8), IFN-γ-AF700 or IFN-γ-APC (Clone B27), TNF-α-PeCy-7 (Clone MAb11), IL-4-PE (Clone 3010.211), IL-2-PE (Clone MQ1-17H12), Gata-3-PeCy-7 (Clone L50-823), all from Becton Dickinson (BD); CD45RA-BV711 (Clone HI100), IL-2-PErCpCy5.5 (Clone MQ1-17H12), IL-5-PE (Clone TRFK5), IL-13-PE (Clone JES10-5A2), T-bet-PerCpCy5.5 (Clone 4B10) were purchased from BioLegend; CD8-Efluor625NC (Clone RPA-T8) from eBioscience; perforin-FITC (Clone B-D48) from Diaclone.

Amelio P., Portevin D., Reither K., Mhimbira F., Mpina M., Tumbo A., Nickel B., Marti H., Knopp S., Ding S., Penn-Nicholson A., Darboe F., Ohmiti K., Scriba T.J., Pantaleo G., Daubenberger C, & Perreau M. (2017). Mixed Th1 and Th2 Mycobacterium tuberculosis-specific CD4 T cell responses in patients with active pulmonary tuberculosis from Tanzania. PLoS Neglected Tropical Diseases, 11(7), e0005817.

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Characterizing CAR-T Cell Activation

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Healthy donor PBMC were activated with 100 U/ml IL-2 and 4 μg/ml PHA and cultured in RPMI media containing 10% FBS (R10) for 72 hrs. After activation, cells were washed, counted and plated at 100,000 cells/well in a 96-well U-bottom plate with 1 x 10⁶ IU/ml of C.Du422.1.LucR virus and cells spinfected for 1 hour at 2500 RPM at 25°C, and then incubated for 7 days in culture. Then, HIV-infected PBMC and donor-matched CAR-T cells were co-cultured for 6 hours in the presence of monensin and brefeldin A to trap the cytokines being produced intracellularly. Each CAR-T cell to target cell condition was setup in triplicates, and then triplicates were pooled for staining at the end of the incubation. At the endpoint, cells were washed, and then stained with live/dead viability dye, CD3-APC, CD4-FITC, and CD8-APC-Cy7 in addition to CD107a-PerCP-Cy5.5 or IL2-PerCP-Cy5.5 and IFN-gamma-Pacific Blue (Biolegend). Data was collected using a BD LSR II flow cytometer (BD Biosciences). FACS data was analyzed to identify the % of untransduced CD8⁺ T cells (UTD) or CAR⁺ CD8⁺ T cells expressing CD107a, IL-2, IFN-gamma, or double-positive IL-2/IFN-gamma expressing cells via FlowJo software (TreeStar). Graphs were generated using GraphPad 7.04.

Anthony-Gonda K., Bardhi A., Ray A., Flerin N., Li M., Chen W., Ochsenbauer C., Kappes J.C., Krueger W., Worden A., Schneider D., Zhu Z., Orentas R., Dimitrov D.S., Goldstein H, & Dropulić B. (2019). Multi-specific anti-HIV duoCAR-T cells display broad antiviral activity and potent elimination of HIV-infected cells in vivo. Science translational medicine, 11(504), eaav5685.

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Phenotypic Analysis of HIV-Specific CD4 T Cells

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Following overnight stimulation as described above, PBMCs (RV21 HIV-infected subjects) were subjected to staining and flow cytometric analysis for measuring de nova frequencies of C. albicans and CMV-specific CD4 T cells. Cells were first stained for viability with LIVE/DEAD Fixable Aqua Blue (Life Technologies), followed by surface staining, including CD3-APC-H7 (BD Bioscience), CD4-PE-Cy5 (BD Bioscience), CD8-BV785 (Biolegend). Cells were then fixed, permeabilized (BD Bioscience) and subjected to intracellular staining, including IL-17-Alexa Fluor 488 (eBioscience), IL-22-APC (eBioscience), IL-2-PerCP-Cy5.5 (Biolegend), IFN-γ-Alexa Fluor 700 (eBioscience), MIP-1β-PE-Cy7 (BD Bioscience). Following staining, cells were acquired using the LSRII Fortessa Analyzer (BD) and data were analyzed using FlowJo (Tree Star). Where appropriate, Boolean gating and Spice analysis were performed using the FlowJo program to analyze the poly-functional characteristics of antigen-specific CD4 T cells.

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Multiparametric Analysis of Tumor-Infiltrating T Cells

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Thawed live cells were stained with and anti-CD103 BV421, anti-CD45 BV510, anti-CD8 BV605, anti-CCR7 BV711, Anti-CD45Ro BV785, anti-CD69 PEcy7, anti-CD4 AF700 and anti-CD19 APC-cy7, anti-CD11c APC/Fire 750 (all from Biolegend), prior to sorting as above. Live melanoma cells (CD45^- SSC-A^high FSC-A^high cells) and three populations of CD8⁺ CD4^- T cells were sorted, the latter as live CD11c^- CD19^- CD45⁺ CD45Ro⁺ CCR7^- lymphocytes distinguished by the marker combination CD69⁺ CD103^-, CD69⁺ CD103⁺ and CD69^- CD103^-.
T cells were stained with anti-PD1(pembrolizumab) or Isotype IgG₄ (BD). T cells and melanoma cells were cultured for 24 hours at a 2:1 effector to target ratio with anti-CD107a-AF488 antibody (Biolegend) or BD GolgiPlug (Brefeldin A) in complete human media (online supplemental methods). For non-sorted samples, 50,000 cells from tumor samples or 25,000 cells from expanded TILs were cocultured.
After 24 hours, cells were restained with all the extracellular markers used for sorting, adding anti-41BB-PercP-Cy5.5 (Biolegend) to anti-CD107a stained cells. Cells incubated with GolgiPlug were permeabilized with eBioscience FoxP3/Transcription Factor Staining Buffer Set (Invitrogen) prior to intracellular staining with anti-IFNγ-PEDazzle594, anti-TNFα-APC, anti-Granzyme B-FITC and IL-2-PercPCy5.5 (all Biolegend).

Pizzolla A., Keam S.P., Vergara I.A., Caramia F., Thio N., Wang M., Kocovski N., Tantalo D., Jabbari J., Au-Yeung G., Sandhu S., Gyorki D.E., Weppler A., Perdicchio M., McArthur G.A., Papenfuss A.T, & Neeson P.J. (2022). Tissue-resident memory T cells from a metastatic vaginal melanoma patient are tumor-responsive T cells and increase after anti-PD-1 treatment. Journal for Immunotherapy of Cancer, 10(5), e004574.

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