Rmil 12

Manufactured by Thermo Fisher Scientific

Sourced in United States

The RmIL-12 is a laboratory instrument designed for the detection and measurement of interleukin-12 (IL-12) in various biological samples. It utilizes a reliable and sensitive immunoassay-based detection method to quantify IL-12 levels accurately.

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12 protocols using rmil 12

NK Cell Functional Assays

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In adoptive transfer setups, total splenocytes were labeled with 0,5 μM CellTrace Violet (eBioscience) during a 10 min incubation at 37 degrees. CTV was quenched by a three-fold washing step in a FCS-supplemented PBS-based buffer. For IFNγ/CD107a functional studies, 3x10⁶ splenocytes were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS (Greiner) in the presence of GolgiStop (BD) and anti-CD107a (BD). Cells were cultured in MaxiSorp flat-bottom plates (Nunc) for 4 hours at 37°C after overnight pre-coating with antibodies (anti-NK1.1 and anti-Ly49D at 10 μg/ml, anti-NKG2D at 5μg/ml) or in presence of cytokine stimulation (rmIL-12 at 25 ng/ml, rmIL-18 at 5 ng/ml and rmIL-2 at 20 ng/ml (all from Peprotech)). For ELISA, 2x10⁴ sort-purified splenic NK cells, isolated from either uninfected or 1,5 days MCMV infected animals, were cultured in tissue culture medium containing RPMI 1640 (Gibco), 10% FCS (Greiner), Glutamax (LifeTechnologies), Gentamicin (Gibco) and β-mercaptoethanol (Cell Culture Core, VIB). Tissue culture medium was supplemented with a low dose of rmIL-15 (10 ng/ml, Peprotech) in presence or absence of cytokine stimulation (rmIL-12 at 25 ng/ml and rmIL-18 at 5 ng/ml, both from Peprotech). After 24 hours, plates were spun down, culture supernatant was collected and stored at minus 20°C.

Vetters J., van Helden M., De Nolf C., Rennen S., Cloots E., Van De Velde E., Fayazpour F., Van Moorleghem J., Vanheerswynghels M., Vergote K., Boon L., Vivier E., Lambrecht B.N, & Janssens S. (2023). Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection. iScience, 26(12), 108570.

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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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Experimental Autoimmune Encephalomyelitis Induction

Cited 3 times

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EAE was induced in Tg4 mice by s.c. injection at the tail base of 100 μl of a sonicated emulsion containing equal volumes of CFA and either 1 mg of spinal cord homogenate suspended in PBS or 200 μg of MBP Ac1-9[4K] in PBS. CFA was supplemented with 4 mg ml⁻¹ heat-killed Mycobacterium tuberculosis (both from Difco). Pertussis toxin (200 ng) (Sigma-Aldrich) was administered i.p. in 500 μl of PBS on days 0 and 2 (refs 17 (link), 57 (link)). Alternatively, EAE was induced by adoptive transfer of MBP Ac1-9-specific Th1 cells. Briefly, Tg4 splenocytes were cultured for 5 days with 10 μg ml⁻¹ MBP Ac1-9[4K] and 5 ng ml⁻¹ rmIL-12 (PeproTech), with 20 U ml⁻¹ rhIL-2 (R&D Systems) added after 48 h. Live in vitro-polarized Th1 cells (1 × 10⁷) were i.v. transferred. Tg4 Rag-1^−/− mice lack nTreg and develop EAE spontaneously19 (link). Individual mice were monitored daily for EAE and scored as follows: 0, no disease; 1, flaccid tail; 2, hindlimb weakness and/or impaired righting; 3, hindlimb paralysis; 4, hind and forelimb paralysis; 5, moribund or dead.

Burton B.R., Britton G.J., Fang H., Verhagen J., Smithers B., Sabatos-Peyton C.A., Carney L.J., Gough J., Strobel S, & Wraith D.C. (2014). Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy. Nature Communications, 5, 4741.

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Th1 Effector Cell Generation from Tg4 Mice

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Th1 effector cells were generated in vitro by culturing splenocytes, from which red blood cells were depleted, from Tg4 mice with 10 μg/ml MBP Ac1-9 [4K] and 5 ng/ml rmIL-12 (Peprotech) for 72 hours in complete RPMI. Cells were further expanded for 6-8 days in complete RPMI supplemented with 20 U/ml rhIL-2 (R&D) (Hill et al., 2013 ). Splenocytes from tolerized mice were also expanded in vitro before analysis by culturing for 5 days in complete RPMI with 10 μg/ml MBP Ac1-9 [4K] and 20 U/ml rhIL-2 (Anderson et al., 2005 (link)). Bone marrow-derived dendritic cells were generated from B10.PL mice (B10.PL-H2^uH2-T18^a/(73NS) SnJ originally from the Jackson Laboratory) by culturing the non-adherent fraction of disaggregated bone marrow with 10 ng/ml rmGM-CSF (Miltenyi Biotec) in complete RPMI for 10-12 days (Inaba et al., 1992 (link)). BM-DC were routinely > 85% MHC-II⁺ as measured by flow cytometry.

Bevington S.L., Ng S.T., Britton G.J., Keane P., Wraith D.C, & Cockerill P.N. (2020). Chromatin Priming Renders T Cell Tolerance-Associated Genes Sensitive to Activation below the Signaling Threshold for Immune Response Genes. Cell Reports, 31(10), 107748.

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Experimental Autoimmune Encephalomyelitis Induction

Cited 3 times

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EAE was induced in Tg4 mice by s.c. injection at the tail base of 100μl of a sonicated emulsion containing equal volumes of CFA and either 1mg of spinal cord homogenate suspended in PBS, or 200μg of MBP Ac1-9[4K] in PBS. CFA was supplemented with 4mg ml⁻¹ heat-killed Mycobacterium Tuberculosis (both from Difco). 200ng of Pertussis toxin (Sigma Aldrich) was administered i.p. in 500μl of PBS on days 0 and 2. ^{17 (link),57 (link)} Alternatively, EAE was induced by adoptive transfer of MBP Ac1-9-specific Th1 cells. Briefly, Tg4 splenocytes were cultured for 5 days with 10μg ml⁻¹ MBP Ac1-9[4K] and 5ng ml⁻¹ rmIL-12 (PeproTech), with 20U ml⁻¹ rhIL-2 (R&D Systems) added after 48 hours. 1×10⁷ live in vitro-polarised Th1 cells were transferred i.v.. Tg4 Rag-1^−/− mice lack nTreg and develop EAE spontaneously^{19 (link)}. Individual mice were monitored daily for EAE and scored as follows: 0, no disease; 1, flaccid tail; 2, hind limb weakness and/or impaired righting; 3, hind limb paralysis; 4, hind and fore limb paralysis; 5, moribund or dead.

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Naïve SMARTA Cell Polarization Protocols

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Naïve SMARTA cells were negatively isolated by using CD4⁺ T cell isolation kit (Miltenyi or StemCell). 2 × 10⁶ SMARTA cells were seeded on 24 well plates coated with 8 µg/mL αCD3 (clone 17A2, BioXcell) and αCD28 (clone 37.51, BioXcell). For T_H1polarization, Smarta cells were given with 20 µg/mL of αIL-4 (clone 11B11, BioXcell) and αTGF-β (clone 1D11, BioXcell) and 20 ng/mL of rmIL-12 (Peprotech). For IL-6 condition, 10 µg/mL of αIFN-γ (clone XMG1.2, BioXcell) and αIL-12 (clone R1-5D9, BioXcell) and 20 ng/mL of rmIL-6 (Peprotech) were added in culture media.

Choi Y.S., Gullicksrud J.A., Xing S., Zeng Z., Shan Q., Li F., Love P.E., Peng W., Xue H.H, & Crotty S. (2015). LEF-1 and TCF-1 orchestrate T follicular helper cell differentiation by regulating differentiation circuits upstream of Bcl6. Nature immunology, 16(9), 980-990.

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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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Experimental Autoimmune Encephalomyelitis Model

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For active EAE, mice were immunized subcutaneously (s.c.) on the back with 200 μg of MOG_35-55 (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (Difco Lab, Detroit, MI) containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco). Two hundred ng of pertussis toxin (List Biological Lab, Epsom, England) was given i.p. on days 0 and 2 post immunization (p.i.). For passive EAE, GFAP-shIFN-γR or GFAP-shVec lentivirus injected mice were transferred with 3.0×10⁷ polarized MOG_35-55-specific Th1 or Th17 cells/mouse 4 hours after sublethal irradiation (550 Rad). To prepare MOG-specific polarized T cell populations, draining lymph nodes and spleen cells were prepared from mice immunized as described above at day 9 p.i. Cells were cultured for 4 days with MOG_35-55 at a concentration of 25 μg/ml under Th1- (20 ng/ml rmIL-12 [PeproTech], 2 μg/ml anti-IL23p19 [eBioscience]) or Th17- (20 ng/ml rmIL-23 [PeproTech]) polarizing conditions (28 (link)). Mice were scored daily for appearance of clinical signs of EAE on a scale from 0 to 5 as described previously (29 (link)): 0, no clinical signs; 1, fully limp tail; 2, paralysis of one hind limb; 3, paralysis of both hind limbs; 4, paralysis of trunk; 5, moribund or death.

Ding X., Yan Y., Li X., Li K., Ciric B., Yang J., Zhang Y., Wu S., Xu H., Chen W., Lovett-Racke A.E., Zhang G.X, & Rostami A. (2015). Silencing IFN-γ binding/signaling in astrocytes vs. microglia leads to opposite effects on CNS autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 194(9), 4251-4264.

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T Cell Activation and Degranulation Assay

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Thawed, washed, and rested lymphocytes were cultured in 96-well U-bottom plates, at a concentration of 2 × 10⁵ cells in a total volume of 200 µl of complete medium, for 18 h (37°C, 5% CO₂) with or without IL-12 plus IL-18 [1 ng/ml recombinant mouse (rm)-IL-12 (PeproTech, Rocky Hill, NJ, USA) plus 20 ng/ml rmIL-18 (MBL, Woburn, MA, USA)] or IL-2 [100 ng/ml rmIL-2 (PeproTech)]. Two microliters of anti-CD107a-FITC were added to each well after 14 h and GolgiPlug (containing Brefeldin A, 1/1,000 final concentration; BD biosciences, Oxford, UK) and GolgiStop (containing Monensin, 1/1,500 concentration; BD biosciences, Oxford, UK) were added after 15 h.

White M.J., Beaver C.M., Goodier M.R., Bottomley C., Nielsen C.M., Wolf A.S., Boldrin L., Whitmore C., Morgan J., Pearce D.J, & Riley E.M. (2017). Calorie Restriction Attenuates Terminal Differentiation of Immune Cells. Frontiers in Immunology, 7, 667.

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Naïve SMARTA Cell Polarization Protocols

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