Inguinal, axillary, brachial, mesenteric, and submandibular lymph nodes (LNs) were excised and T-cells isolated as previously described (Tremblay et al., 2017). Brie y, CD8 + CTLs were enriched by panning and cultured at a density of 1 x 10 6 cells/mL in complete RPMI (cRPMI) media supplemented with CD28 (1 µg/mL; clone 37.51, Tonbo Biosciences, San Diego, CA, USA), gentamycin (5 µg/mL, Gibco), IL-2 (100 U/mL, Peprotech, Rocky Hill NJ, UA), IL-12 (1 ng/mL, MilliporeSigma) in culture dishes that had been precoated overnight with 1 µg/mL CD3 (clone 145-2C11, Tonbo Biosciences) in phosphate buffered saline (PBS, 1X concentration; Corning). cRPMI media was a solution of RPMI 1640 (MilliporeSigma) with 10% FBessence, 100 U/mL of Penicillin/Streptomycin, 55 µM of b-mercaptoethanol, and 10 mM HEPES. Cells were maintained with fresh cRPMI media supplemented with IL-2 (100 U/mL) as required over 7 days. In preparation for labelling, cells were collected from culture dishes, washed with PBS, and re-suspended in cRPMI without antibiotics.
Il 12
Manufactured by Merck Group
Sourced in United States
IL-12 is a laboratory reagent that functions as a cytokine. It is involved in the regulation of immune responses and plays a role in the differentiation of T helper cells. The detailed specifications and intended use of this product are not available for inclusion in this response.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (4 mentions)
Interleukin 4 (il 4), by Merck Group (4 mentions)
Interleukin 6 (il 6), by Merck Group (3 mentions)
Il 1β, by Merck Group (3 mentions)
Gentamycin, by Thermo Fisher Scientific (2 mentions)
Anti ifn γ, by Merck Group (2 mentions)
Tgf β, by Merck Group (2 mentions)
Il 23, by Merck Group (2 mentions)
Anti il 4, by Merck Group (2 mentions)
Immunocult human cd3 cd28 t cell activator, by STEMCELL (2 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Clone 37, by Cytek Biosciences (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Il 13, by Merck Group (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
14 protocols using il 12
1
Isolation and Culture of CD8+ CTLs
2
Dolphin Lymphocyte Immune Responses
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In order to assess ability of dolphin lymphocytes to respond to a Th1, Th2, or Treg stimulus, dolphin PBMCs (2 × 106 cells/ml) were incubated in 96 well flat bottom plates with human recombinant cytokines at concentrations of 0 (unstimulated), 1, 10, and 25 pg/ml for 24 h. To assess a Th1 response, cells were stimulated with IL-12 (Millipore Sigma, Burlington, MA 01803, USA) and IFNγ (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and analyzed for IFNγ expression. To stimulate a Th2 response, cells were stimulated with IL-4 (Millipore Sigma, Burlington, MA 01803, USA) and analyzed for IL-4 and IL-13 expression. To assess a Treg response, cells were stimulated with IL-2 (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and TGFβ (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and analyzed for TGFβ and IL-10 expression. After a 24 h incubation at 37°C and 5% CO2, cells were collected from the plates, centrifuged at 220 g for 10 min, re-suspended in RNAlater solution (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and stored at 4°C for up to 1 month. RNAlater samples were then moved to −20°C for long-term storage.
3
Polarization of Naïve CD4+ T Cells
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Naïve CD4+ T cells were separated from PBMC samples using naïve CD4+ T cell Isolation kit (Miltenyi, Germany) according to the kit’s protocol. The cells were activated with anti-CD28 (2 μg/mL, eBioscience, USA) and anti-CD3 (5 μg/mL, eBioscience, USA). The polarization of Th1/Th2/Th17 was performed for 3 days in the presence of different polarizing conditions (25 (link), 26 (link)). For Th1 polarization, anti–IL-4 (10 μg/ml, eBioscience, USA) and IL-12 (10 ng/mL, Sigma, USA) were applied. For Th2 polarization, IL-4 (2.5 ng/mL, Sigma, USA) and anti–IFN-γ (10 μg/mL, eBioscience, USA) were applied. For Th17 polarization, TGF-β (5 ng/mL, Sigma, USA), IL-6 (10 ng/mL, Sigma, USA), IL-1β (10 ng/mL, Sigma, USA), IL-23 (20 ng/ml, Sigma, USA), anti–IFN-γ (10 μg/ml), and anti–IL-4 (10 μg/ml) were applied. Naïve CD4+ T cells were maintained in RPMI 1640 medium (HyClone, USA) supplemented with 10% FBS (HyClone, USA) at 37°C and 5% CO2 for all experiments. The expression of MALT1 in polarized cells was analyzed, and the naïve CD4+ T cells being cultured normally for 3 days were utilized as control.
4
T Helper Cell Polarization Protocol
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The polarization of Th1, T-helper 2 (Th2), and Th17 cells was performed as described previously (21 (link), 22 (link)). Briefly, transfected naïve CD4+ T cells were stimulated with the following polarizing conditions for 3 days: IL-12 (10 ng/ml, Sigma, USA) and anti–IL-4 (5 μg/ml, Affinity, USA) for Th1 polarization; IL-4 (2.5 ng/ml, Sigma, USA) and anti-IFN-γ (5 μg/ml, Affinity, USA) for Th2 polarization; TGF-β (10 ng/ml, Sigma, USA), IL-6 (10 ng/ml, Sigma, USA), IL-1β (10 ng/ml, Sigma, USA), IL-23 (10 ng/ml, Sigma, USA), anti–IL-4 (5 μg/ml), and anti-IFN-γ (5 μg/ml) for Th17 polarization. After polarization, the cells and supernatants were collected for flow cytometry and ELISA, respectively.
5
Modulation of NK Cell Cytokine Release by IL-33 Blocking Antibodies
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IL33 amplifies both TH1 and TH2 responses by activating different leukocytes, in particular NK cells [36] (link). Human PBMCs were isolated from healthy blood donors by Ficoll gradient. The blood sampling of healthy volunteers was approved by the local ethics committee (Bayerische Landesärztekammer, Munich) and subjects gave written, informed consent. The NK cells were purified from PBMC using the negative NK cell isolation kit (Miltenyi Biotec). The average purity was over 96%. For assessment of the IL1RL1-blocking functionality of the monoclonal rabbit antibodies, 1×105 NK cells/well were pretreated with the rabbit antibodies or the respective isotype control antibodies at different concentrations, seeded into a 96-well flat bottom plate and incubated for 1 h at 37°C. NK cells were then stimulated with 10 ng/ml IL-33 (Peprotech) and 1 ng/ml IL-12 (Sigma-Aldrich) and incubated for 20 h in RPMI 1640 medium supplemented with 10% FCS, 1% sodium pyruvate and L-Glutamine, as well as 0.1% 2-Mercaptoethanol. After this, supernatants were harvested, centrifuged and tested for IFN-γ production. For IFN-γ quantification an in-house established ELISA or BD’s CBA flex set platform was used (human IFN-γ flex set, BD Biosciences) according to the manufacturer’s instructions using a FACS Canto II.
6
Inflammatory Cytokine Quantification
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The brain tissues were homogenized, and the protein concentration was determined by BCA kits. IL‐6 (RAB0309), TNF‐α (RAB0477), IL‐1β (RAB0275), IL‐12 (RAB0255), IL‐4 (RAB0300) and IL‐10 (RAB0245) commercial kits were provided by Sigma. The contents of inflammatory cytokines were measured according to the manufacturer's instructions.
7
Priming Cytotoxic T Cells for Adoptive Transfer
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One week prior to cell injection, inguinal, axial, brachial, and mesenteric LNs were collected from treatment- and disease-matched B6-GFP+ mice. Cytotoxic T lymphocytes (CTLs) were isolated as previously described.27 Briefly, CD8+ lymphocytes were enriched using a panning method. The enriched CTL population was cultured in vitro at a density of 1 × 106 cells/mL in complete RPMI media (cRPMI) supplemented with mouse CD28 (1 µg/mL; clone 37.51; eBiosciences), gentamycin (5 µg/mL; Gibco), IL-2 (100 U/mL; Sigma-Aldrich), and IL-12 (100 ng/mL; Sigma-Aldrich) on a coated CD3e (2 µg/mL; clone 145–2C11; Tonbo Biosciences) culture flask to encourage cellular proliferation and activation. Five days before ACT, spleens were isolated from disease- and treatment-matched B6 mice and cultured in vitro in cDMEM media at 37°C in an incubator to act as antigen-presenting cells to the CTLs. Three days before ACT, splenocyte proliferation was prohibited using mitomycin-c (50 µg/mL; Sigma-Aldrich) and after washing, added to the CTL culture at a ratio of 1:10 APC:CTL. R9F (10 µg/mL in DMSO; Genscript) was added to encourage further priming. Fresh cRPMI supplemented with 100 U/mL of IL-2 was added and replaced as needed to maintain the CTLs in culture.
8
Characterization of NK Cell Subsets
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Degranulation and cytokine production of NK cell subsets were determined after cytokine stimulation o/n with IL-12 (10 ng/mL, Sigma Aldrich, St. Louis, MO, USA) and IL-18 (5 ng/mL, MBL, Woburn, MA, USA), after CD16-crosslinking or after co-culture with the cell lines K562, Huh7 and HepG2 and autologous CD8+ T cells (pre-activated with ImmunoCultTM Human CD3/CD28 T Cell Activator (Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions). More detailed information is provided in the supplementary information .
9
Multicolor Flow Cytometric Analysis of Immune Cell Activation
10
Cytokine Profiles in Trichomoniasis
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The pro-inflammatory cytokines, IL-8 and IL-12, were purchased from Sigma Aldrich ® (USA); the protocol of detection was sandwich ELISA, according to the manufacturer's protocol. Samples were screened in a 96-well plate for pretested women; 18 samples of IgM and 16 of IgG for trichomoniasis. Plates were read at 450 nm by ELISA reader [19] . The control group included 15 serum samples of Ali Iraqi Journal of Science, 2021, Vol. 62, No. 2, pp: 449-454 451 apparently healthy women, with no known illness, and screened as above for IL-8 and IL-12 comparison. Exclusion criteria Samples from men, single women, and women under 18 years old were excluded in this study. Only married women with positive anti-T vaginalis IgM or IgG, in comparison with control groups, were involved.
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