Human melanoma MMAC-SF (RRID: CVCL_1420) and WM-115 (RRID: CVCL_0040) cells were propagated in DMEM medium and MEM medium at humid atmosphere (5% CO2, 37°C), respectively. The culture medium was supplemented with 10% FBS, penicillin (100 u/mL) and streptomycin (100 mg/mL). FITC Annexin V Apoptosis Detection Kit I (cat# 556547) was supplied by BD Biosciences (San Jose, CA, USA). Chromatin Immunoprecipitation Kit (17–10086) was obtained from Merck (Millipore, CA, USA). H3K4Me3 (9751S) was purchased from Cell Signaling Technology (Danvers, MA, USA). IFN-γ (300–02) was supplied by Peprotech (Jiangsu China). PE anti-mouse PD-L1 (124307) and PE anti-mouse PD-1 (135205) were purchased from Biolegend (San Diego, CA, USA). Anti PD-L1 antibody (ab205921) was obtained from Abcam (Cambridge, MA, USA). Anti DPY30 antibody (MA5-32900) was acquired Invitrogen (Carlsbad, CA, USA).
Ifn γ 300 02
Manufactured by Thermo Fisher Scientific
Sourced in China
IFN-γ (300–02) is a laboratory reagent produced by Thermo Fisher Scientific. It is a recombinant human interferon gamma, a signaling protein involved in the immune response. The core function of this product is to serve as a research tool for studying interferon gamma and its role in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α 300 01a, by Thermo Fisher Scientific (2 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Ab125066, by Abcam (1 mentions)
Ab91654, by Abcam (1 mentions)
Ab216876, by Abcam (1 mentions)
Ferrostatin 1, by Selleck Chemicals (1 mentions)
Ab8227, by Abcam (1 mentions)
Necrosulfonamide, by Merck Group (1 mentions)
Gsk 872, by Selleck Chemicals (1 mentions)
Ac yvad cmk sml0429, by Merck Group (1 mentions)
Hmgb1 1690 hmb, by R&D Systems (1 mentions)
Flagellin, by InvivoGen (1 mentions)
Adenosine triphosphate (atp), by Selleck Chemicals (1 mentions)
Upper chamber, by Corning (1 mentions)
Lipopolysaccharides (lps), by Solarbio (1 mentions)
M csf 300 25, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Thp 1 cells, by National Collection of Authenticated Cell Cultures (1 mentions)
8 protocols using ifn γ 300 02
1
Apoptosis and Epigenetic Regulation in Melanoma
2
Antibodies and Ferroptosis Inducers Protocol
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Antibodies against CRTC3 (ab91654, WB, 1:1000), SLC7A11 (ab216876, WB, 1:1000), GPx4 (ab125066, WB 1:1000, IHC 1:100), and β-actin (ab8227, WB, 1:1000) were purchased from Abcam. Sorafenib (S7397), erastin (S7242), RSL3 (S8155) and Ferrostatin-1 (S7243) were purchased from Selleck Chemicals. PRGL493 (HY-139180) was purchased from MedChemExpress. IFN-γ (300-02) was purchased from PEPROTECH.
3
Immune Signaling Pathway Modulation
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Pam3CSK4 (tlrl-pms), Poly(I:C) (tlrl-piclv), LPS (tlrl-peklps) and Flagellin (tlrl-pafla) were purchased from Invivogen. TNF-α (300–01A), IFN-γ (300–02) and IL-1α (200–01A) were purchased from Peprotech. HMGB1 (1690-HMB) was from R&D system. Rapamycin (S1039), Torin-1 (S2827), z-DEVD-FMK (S7312), z-VAD-FMK (S7023), GSK’872 (S8465) and ATP (S5260) were purchased from Selleck. Ac-YVAD-CMK (SML0429) and necrosulfonamide (480,073) were purchased from Sigma.
4
M1 Macrophage Migration Assay
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Phorbol 12-myristate 13-acetate (PMA; P6741, Solarbio, China) was used to differentiate THP-1 cells into macrophages. Macrophages were polarized to M1 macrophages by incubation with IFN-γ (30002, PeproTech, China) and LPS (L8880, Solarbio, China) for 24 h. M1 macrophages without serum were seeded in the upper chamber (Corning, NY, USA). The U87 cells (1.0×105) were cultured with 10% FBS in DMEM at the bottom plate. After 48 h incubation, the migrated M1 macrophages were counted in the bottom chamber. Each experiment was performed three times.
5
Differentiation of THP-1 Macrophages
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Human monocytic THP-1 cells were purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in complete RPMI-1640 medium containing 10% fetal bovine serum, 10 mM glucose, 2 mM L-glutamine and 100 U mL−1 penicillin-streptomycin. THP-1 cells were differentiated into M0 macrophages by incubation with 100 ng mL−1 phorbol 12-myristate 13-acetate (PMA) (P8139, Sigma-Aldrich) for 72 h. Once the cells were adherent, they were transferred to PMA-free media to obtain resting macrophages. These cells were incubated with 20 ng mL−1 human macrophage colony-stimulating factor (M-CSF) (300-25, PeproTech) and then co-stimulated with 100 ng mL−1 LPS (L2630, Sigma-Aldrich) plus 20 ng mL−1 IFN-γ (300-02, PeproTech) or stimulated with 10 ng mL−1 IL-4 (200-04, PeproTech) for 24 h to generating inflammatory or anti-inflammatory macrophages.
6
Cytokine Effects on Pancreatic Stellate Cells
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To determine the effects of the inflammatory reactions present in the diabetic pancreas on the PSC’s cytokine expression, isolated PSCs in passage 3–8 were either untreated or cultured for 24 h in the presence of one of the following cytokines: 50 U/mL interleukin-1β (IL-1β; 200-01; PeproTech, Rocky Hill, NJ, USA), 20 ng/mL tumor necrosis factor-α (TNF-α; 310-01A, PeproTech), 10 ng/mL interleukin-6 (IL-6; 216-16, PeproTech), 1,000 U/mL interferon-γ (IFN-γ; 300-02, PeproTech). Then the culture medium was collected, and its cytokine contents were analyzed according to the manufacturer’s instructions for Mouse ProInflammatory 7-Plex Assay Ultra-Sensitive Kit (K15012C-1, Meso Scale Diagnostics). This allowed us to assess the concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin 12p70 (IL-12p70), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and mammalian keratinocyte chemoattractant (mKC).
7
Macrophage Polarization and Cytokine Secretion
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Monocyte-derived macrophages were stimulated for 24 h at 37 °C with 10 ng/mL LPS (E.Coli 0111:B4, L2630, Sigma-Aldrich) and 20 ng/mL IFN-γ (300-02, PeproTech) or 50 ng/mL IL-4 (200-04, PeproTech). In some cases, specific inhibitors and blockers were used including 10 nM UNC-569 (445835-10MG, Millipore, Billerica, USA), 10 μg/mL anti-anxA1 (clone 1B; produced in house) or 10 μg/mL isotype control (14-4714-85, eBioscience, San Diego, USA). Neutrophil MVs were also added at the indicated concentrations. Supernatants were collected for Cytometric Bead Array for IL-12p70, IL-1β, IL-10 and TGF-β (558264, BD Biosciences, San Jose, USA) following manufacturer's instructions. Cells were detached, blocked in 160 μg/mL human IgG (G4386, Sigma-Aldrich) at 4 °C for 15 min, and labelled with 1.25 μg/mL anti-HLADR/DP/DQ-FITC, 1 μg/mL anti-CD86-PE, and 4 μg/mL anti-CD206 antibodies at 4 °C for 30 min. Cells were acquired on a LSRFortessa cytometer.
8
Immunomodulatory effects of MSC-Ex
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HaCaT (human keratinocyte) cells were maintained in DMEM (Invitrogen-Gibco, Carlsbad, CA) containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml) (Invitrogen-Gibco) at 37 °C in a humidified 5% CO2 incubator. HaCaT cells were pretreated with bacterial LPS (E. coli, serotype 055:B5, 100 μg/ml; Sigma-Aldrich, St. Louis, MO) for 4 h or TNF-α (300-01 A; PEPROTECH, Rocky Hill, NJ) and IFN-γ (300-02; PEPROTECH) (10 ng/ml each) for 2 h and then treated with MSC-Ex (30 μg/ml) in the presence of LPS or TNF-α/IFN-γ for an additional 24 h. RAW264.7 cells were incubated with LPS (1 µg/ml) for 4 h. After incubation, the RAW264.7 cells were washed three times in ice-cold UC-MSCs were washed twice with cold phosphate-buffered saline (PBS), and treated with MSC-Ex (30 μg/ml) for 24 h.
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