For immunohistochemical analysis, the dewaxed sections were boiled in 10 mmol/L citrate buffer for 20 min for epitope retrieval following washing three times in PBS. After incubating in 3% hydrogen peroxide, the sections were blocked with 1% BSA for 1 hour. The slices were incubated with anti-rat antibodies against IL-10 (1:200, Servicebio, China), TGF-β3 (1:200, Servicebio, China), IL-6 (1:400, Servicebio, China), TNF-α (1:300, Servicebio, China), IFN-γ (1:150, Servicebio, China), α-SMA (1:1000, Servicebio, China), MCP-1 (1:500, Servicebio, China), CCR2 (1:800, Servicebio, China) and CCRL2 (1:200, Servicebio, China) at 4°C overnight and then incubated with HRP labeled Goat anti-rabbit IgG general secondary antibody (DAKO, Denmark). Finally, the antibody binding was visualized by using a DAB kit (Thermo Scientific). All sections were imaged using an automatic slide scanning system (AxioScan.Z1, Zeiss, Germany) at 20X magnification.
Ifn γ
Manufactured by Wuhan Servicebio Technology
Sourced in China
IFN-γ is an immune system protein that plays a crucial role in the body's defense against infections and diseases. It is produced by various cells, including T cells and natural killer cells, and is involved in the regulation of immune responses. IFN-γ helps to activate and coordinate the functions of the immune system, making it a valuable tool for researchers and clinicians.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Wuhan Servicebio Technology (1 mentions)
Interleukin 6 (il 6), by Wuhan Servicebio Technology (1 mentions)
Dab kit, by Thermo Fisher Scientific (1 mentions)
Mcp 1, by Wuhan Servicebio Technology (1 mentions)
α sma, by Wuhan Servicebio Technology (1 mentions)
Rhil 4, by R&D Systems (1 mentions)
Recombinant human gm csf rhgm csf, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
3 protocols using ifn γ
1
Immunohistochemical Analysis of Cytokines and Chemokines
2
Cytokine profiling of immune cell cultures
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In this study, HOK cells, Epi1.2 cells, T cells, and Epi1.2 + T cells were cultivated in 48-well plates for 24 h. Subsequently, the supernatant from each group was collected and used to detect various cytokines, including interleukin (IL)-6 (Servicebio, GEH0001), IL-1β (Servicebio, GEH0002), IL-17 (MultiSciences; EK117/2–96), transforming growth factor beta (TGF-β) (MultiSciences, EK981-96), tumor necrosis factor alpha (TNF-α) (Servicebio, GEH0004), and interferon gamma (IFN-γ) (Servicebio, GEH0006). The quantification of these cytokines was performed using ELISA kits according to the manufacturer’s guidelines. The absorbance at 450 nm was measured using the microplate reader.
3
Generation of CIKs and DCs for Anti-Tumor Immunotherapy
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Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors by density gradient centrifugation, and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) containing 10% FBS and penicillin/streptomycin. Following 4 h of culture, the suspended cells (T cells) were grown to generate CIK cells in RPMI-1640 with 10% FBS containing 500 ng/mL anti-CD3 antibody, 100 U/mL IFN-γ (Servicebio, Wuhan, China) and 10 μg/mL polyhydroxyalkanoates (Solarbio, Beijing, China). In addition, adhered cells were used for dendritic cell differentiation via culturing in RPMI supplemented with 1000 U/mL recombinant human GM-CSF (rhGM-CSF; R&D, MN, US) and 500 U/mL rhIL-4 (R&D, MN, US) for 7 days. Next, A549 and HepG2 cells were lysed (Repeated freeze-thaw procedure), and the supernatant was obtained as the tumor antigen. On day 8, the supernatant and 10 ng/mL TNF-α and 10 ng/mL IL-1β were added to the DCs medium, after which the culture was maintained for two more days. Then, DCs and CIKs were co-cultured at a ratio of 1:10 for two days. On day 12, we added the Nb36 to DC-CIK cells to mediate CTLA-4 blockade. The study were approved by the local ethics committee of Hainan Medical University.
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