Rmil 15

Manufactured by Thermo Fisher Scientific

Sourced in United States

The RmIL-15 is a laboratory equipment product designed for use in scientific research applications. It serves as a tool for the measurement and analysis of interleukin-15 (IL-15), a cytokine involved in various biological processes. The core function of the RmIL-15 is to provide researchers with a reliable and accurate means of quantifying IL-15 levels in samples.

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Lab products found in correlation

Golgistop, by BD (3 mentions) Rmil 12, by Thermo Fisher Scientific (3 mentions) Rmil 18, by R&D Systems (2 mentions) Rmil 7, by Thermo Fisher Scientific (2 mentions) Rmil 10, by Thermo Fisher Scientific (1 mentions) Rmtgf β, by R&D Systems (1 mentions) Shp099, by Selleck Chemicals (1 mentions) Non tc treated culture dishes, by Corning (1 mentions) Torin 2, by Bio-Techne (1 mentions) Anti nk1.1 pk136, by BioXCell (1 mentions) 7 aad, by Thermo Fisher Scientific (1 mentions) Azd2014, by Selleck Chemicals (1 mentions) Iscript synthesis kit, by Bio-Rad (1 mentions) Rnaeasy micro kit, by Qiagen (1 mentions) Rhil 2, by R&D Systems (1 mentions) Anti p s6 s235 236 d57.2.2e, by Cell Signaling Technology (1 mentions) Anti cd3ε 145 2c11, by BioLegend (1 mentions) Rmil 2, by R&D Systems (1 mentions) Anti cd8a ly 2 microbeads, by Miltenyi Biotec (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions)

15 protocols using rmil 15

Modulation of NK Cell Responsiveness

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To study the responsiveness of NK cells to IL-15, splenocytes were resuspended with RPMI 1640 medium and plated on 24-well plates. Then splenocytes were treated with rmIL-15 (Peprotech, 100 ng/mL) for 1, 2, 3, 5, 7, or 9 days, followed by flow cytometry. To investigate the influence of cytokines on METTL3 expression in NK cells, we treated splenocytes with rmIL-17F (25 ng/mL, Peprotech), rmIL-27 (25 ng/mL, Peprotech), rmIL-10 (25 ng/mL, Peprotech), rmIL-15 (25 ng/mL, Peprotech), rmTGF-β (25 ng/mL, R&D) and rmIL-18 (25 ng/mL, R&D) for 3 days. To study the effect of MC38 cells on METTL3 expression in NK cells, purified NK cells were cocultured with MC38 cells under the stimulation of rmIL-15 (10 ng/mL) with the presence or absence of SIS3 (20 μM, APExBIO, Houston, TX, USA) or GW788388 (20 μM, APExBIO) for 2 days. To observe the effects of SHP-2 on the NK cells, splenocytes were treated with rmIL-15 (50 ng/mL, Peprotech) with or without SHP-2 inhibitor SHP099 (4 μM, Selleck, Houston, TX, USA) for 3 days.

Song H., Song J., Cheng M., Zheng M., Wang T., Tian S., Flavell R.A., Zhu S., Li H.B., Ding C., Wei H., Sun R., Peng H, & Tian Z. (2021). METTL3-mediated m6A RNA methylation promotes the anti-tumour immunity of natural killer cells. Nature Communications, 12, 5522.

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Cytokine-Induced NK Cell Activation Assay

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Splenic lymphocytes were prepared and cultured with cytokines (rmIL-15 100ng/ml; rmIL-12 25ng/ml from Peprotech and rmIL-18 5ng/ml from PBL), or on antibody coated plates (anti-NKp46, anti-NK1.1, anti-Ly49D all at 10μg/ml on Immulon 2HB plates) and Golgi-stop (4μl in 6ml; BD-Biosciences) in the presence of anti-CD107a for 4h. Surface and intracellular stainings were then performed and IFN-γ production as well as CD107a exposure was measured by flow cytometry.

Fauteux S., Faure F., Marotel M., Geary C., Daussy C., Sun J.C, & Walzer T. (2019). Styk1 expression is a hallmark of murine NK cells and other NK1.1+ subsets but is dispensable for NK cell development and effector functions. European journal of immunology, 49(5), 677-685.

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Generating Bone Marrow-Derived Myeloid Cells

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To generate bone marrow derived macrophages and cDC1, bone marrow leukocytes were resuspended at 1.5×10⁷ cells/10mL in DC media (RMPI 1640 + 25mM HEPES + 10% FBS, 1% L-glutamine, 1% 200mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin-streptomycin, 0.5% sodium bicarbonate, 0.01% 55 mM 2-mercaptoethanol supplemented with 200 ng/mL FLT3-L and 50 ng/mL GMCSF) or macrophage media (DMEM + 10%FBS, 1% L-glutamine, 1% penicillin-streptomycin, 0.5% sodium pyruvate, supplemented with 50 ng/ml M-CSF and then plated in 10cm non-TC treated culture dishes (Corning). Bone marrow leukocytes were then cultured for 9 days in DC media with an additional 5 mL of DC media added on D5. cDCP were either harvested on D9 or media was changed and BM-cDC1 were used for experiments on D15 as described previously (Mayer et al., 2014 (link)). BMDM are cultured for 7 days in macrophage media, with a media change on D3. Isolated ILC1 and NK cells were cultured in CR-10 (RMPI 1640 + 25 mM HEPES + 10% FBS, 1% L-glutamine, 1% 200 mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin-streptomycin, 0.5% sodium bicarbonate, 0.01% 55 mM 2-mercaptoethanol) supplemented with 50ng rmIL-15 (Peprotech). Purified ILC1’s and NK cells were cultured 16 hours prior to electroporation in TC treated plates at ~5 × 10⁵ cells / cm².

Riggan L., Hildreth A.D., Rolot M., Wong Y.Y., Satyadi W., Sun R., Huerta C, & O’Sullivan T.E. (2020). CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells. Cell reports, 31(7), 107651.

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NK Cell Activation and Signaling Assays

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1.5 × 10⁶ splenocytes were cultured on antibody coated plates (anti-NKp46 (Goat polyclonal, R&D), anti-NK1.1 (PK136, BioXCell) at 10 µg/ml on Immulon 2HB or Nunclon plates) with Golgi-stop (BD Biosciences) in the presence of anti-CD107a for 4 hr. Cytokines and mTOR inhibitors were used at the following concentrations unless otherwise stated: rmIL-15 (Peprotech; 100 ng/ml), IL-2 (muIL-2 supernatant; 200 U/ml), Rapamycin (Calbiochem; 25 nM), KU-0063794 (Stemgent; 3 µM), AZD2014 (Selleckchem; 5 µM) and Torin2 (Tocris; 250 nM). Surface and intracellular stainings were then performed and IFN-γ production as well as CD107a exposure was measured by flow cytometry. In some experiments, cell viability was determined using 7AAD (Invitrogen, 250 nM).
For phospho-flow stainings following short-term NK1.1 stimulation, 3 × 10⁶ splenocytes were stimulated using biotinylated NK1.1 (PK136, 5 µg/ml) followed 1 min 30 s later by streptavidin (Life Technologies, 10 µg/ml) and fixed by addition of 10 volumes of Lyse/Fix at the indicated time point.

Marçais A., Marotel M., Degouve S., Koenig A., Fauteux-Daniel S., Drouillard A., Schlums H., Viel S., Besson L., Allatif O., Bléry M., Vivier E., Bryceson Y., Thaunat O, & Walzer T. (2017). High mTOR activity is a hallmark of reactive natural killer cells and amplifies early signaling through activating receptors. eLife, 6, e26423.

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In vitro Expansion and Activation of NK Cells

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Purified NK cells were resuspended with RPMI 1640 complete medium and plated on a 96-well plate (5 × 10⁵ per 200 μL per well). NK cells were treated with rmIL-15 (50 ng/mL; Peprotech,) for 9 days. The concentration of IFN-γ and GzmB in the NK-cell culture supernatant was measured by ELISA kits (Multisciences, Hangzhou, China, Cat # EK280/3-96 for IFN-γ, Cat # EK2173-96 for Gzm B) according to manufacturer instructions.

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NK-cell Stimulation and Transcriptional Analysis

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NK-cell stimulations were performed in the presence of 5,000 U ml⁻¹ rhIL-2, 5 ng ml⁻¹ recombinant murine IL-12 (rmIL-12) (R&D) and 50 ng ml⁻¹ rmIL-15 (PeproTech). RNA was extracted with the Qiagen RNAeasy microkit (Qiagen) and complementary DNA prepared with the iScript synthesis kit (BioRad). Real-time PCR was performed with the following primers: GrzmB-fw 5′-CCAATCAGATATGTGCGGG-3′, GrzmB-rev 5′-GGAAACTATGCCTGCAGCC-3′, Prf-fw 5′-GATGTGAACCCTAGGCCAGA-3′ Prf-rev 5′-GGTTTTTGTACCAGGCGAAA-3′ and the housekeeping gene Rplp0-fw 5′-CCTGGCATTGTCTGTGGAGAC-3′, Rplp0-rev 5′-GCTTCAGCTTTGGCAGGG-3′.

Bottos A., Gotthardt D., Gill J.W., Gattelli A., Frei A., Tzankov A., Sexl V., Wodnar-Filipowicz A, & Hynes N.E. (2016). Decreased NK-cell tumour immunosurveillance consequent to JAK inhibition enhances metastasis in breast cancer models. Nature Communications, 7, 12258.

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Evaluating Immune Cell Signaling Pathways

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For all signaling assays, non-sorted splenocytes or lymph node cells were serum-starved for 1 h at 37 °C before stimulation in complete media without serum. For evaluation of TCR signaling, splenocytes and lymphocytes were stimulated with 20 μg/mL anti-CD3ε (145-2C11; Biolegend) and 50 µg/mL anti-IgG (polyclonal, α-Armenian hamster, Jackson ImmunoResearch) for the indicated times^{69 (link),70 (link)}. For evaluation of pSTAT5 signaling, cells were stimulated for 15 min with recombinant human IL-2 (rhIL-2; NIH AIDS Reagent Program) at indicated concentrations, recombinant mouse IL-15 (rmIL-15; Peprotech) at indicated concentrations, or 10 ng/mL recombinant IL-7 (rmIL-7; Peprotech). Immediately following stimulation cells were fixed with a final concentration of 1.5% methanol-free formaldehyde (Fisher Scientific; Cat. PI28906) for 15 min, and permeabilized with 500 µL of ice-cold methanol per 1 × 10⁶ cells for 30 min^{69 (link),71 (link)}. Cells were washed twice with PBS/2%FBS (Omega Scientific) then stained for 60 min in anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-FoxP3 (FJK-16s), anti-pSTAT5 (Y694; C71E5; Cell Signaling Technologies) or anti-pS6 (S235/236; D57.2.2E; Cell Signaling Technologies), and, for exclusion of non-relevant cells, anti-CD45R (B220; RA3-6B2), anti-CD11b (MI/70), anti-CD11c (N418), and anti-Ly-6G (Gr-1; RB6-8C5).

Mullins G.N., Valentine K.M., Al-Kuhlani M., Davini D., Jensen K.D, & Hoyer K.K. (2020). T cell signaling and Treg dysfunction correlate to disease kinetics in IL-2Rα-KO autoimmune mice. Scientific Reports, 10, 21994.

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Isolation and Activation of CD8+ T Cells

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CD8⁺ cells were enriched from spleen cell preparations via positive selection using anti-CD8a (Ly-2) microbeads (Miltenyi Biotech) and then CD45.2⁺CD3⁺CD8⁺ T cells were sorted by flow cytometry. 5x10⁴ cells were seeded in wells of a 96-well plate with 100μL RPMI 1640 medium supplemented with 1mM sodium pyruvate, 10mM HEPES, 50μM mercaptoethanol, and 10% FCS with 40ng/mL rmIL-15 (Peprotech). Alternatively, wells were coated with anti-CD3 (5μg/mL, clone 145-2C11) and anti-CD28 antibodies (1μg/mL, clone 37.51) at 4°C overnight and then cells were added with 20ng/mL rmIL-2 (R&D systems) for 2 or 3d.

Ushiki T., Huntington N.D., Glaser S.P., Kiu H., Georgiou A., Zhang J.G., Metcalf D., Nicola N.A., Roberts A.W, & Alexander W.S. (2016). Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells. PLoS ONE, 11(9), e0162111.

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Purification and Activation of NK Cells

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Magnetic beads (CD3 Microbead kit, Pan T Cell Isolation kit II, NK cell isolation kit II; Miltenyi Biotec) were used to deplete T cells in BMCs and to purify donor T cells and donor NK cells from spleens (purity > 90%). Where indicated, NK cells were stimulated for 16– 18 h with 2000 IU/mL rhIL‐2 (Proleukin, Novartis), 10 ng/mL rmIL‐15 (PeproTech), or 10 ng/mL rmIL‐12 (PeproTech) + 10 ng/mL IL‐15 + 50 ng/mL rmIL‐18 (MBL) or for 7 days with 2000 IU/mL rhIL‐2. We found that it is important to wash NK cells thoroughly after preactivation, that is at least three times with a large volume of PBS. LPS‐matured BMDCs were generated as previously described 22. ConA blasts were generated by culturing single cell suspensions from BALB/c spleens for 30 min at 37°C, then collecting nonadherent cells. Nonadherent cells were cultured with 5 μg/mL ConA (Sigma) for 48–72 h before used.

Hüber C.M., Doisne J, & Colucci F. (2015). IL‐12/15/18‐preactivated NK cells suppress GvHD in a mouse model of mismatched hematopoietic cell transplantation. European Journal of Immunology, 45(6), 1727-1735.

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Cytotoxicity Assay with YAC-1 Cells

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YAC-1 cells were labeled with CTV (Thermo Fisher Scientific) as described above. Splenocytes were stimulated with recombinant mouse (rm) IL-12 (20 ng/mL, Peprotech), rmIL-15 (20 ng/mL, Peprotech) and rmIL-18 (10 ng/mL, R&D, Minneapolis, MN, USA) for 18 h. Then, the stimulated splenocytes (5 × 10⁶) were cocultured with CTV-labeled YAC-1 cells (5 × 10⁴) for 4 h, followed by an analysis of the BD flow cytometer.

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