Spleens were extracted from mice, homogenized, and treated with ACK RBC lysis buffer to collect mouse splenocytes. Cells were then sorted by FACS using a FACSMelody™ (BD) with antibodies against CD45 (30-F11), CD3Ɛ (145–2C11), CD4 (GK1.5), CD8α (53–6.7), and NK1.1 (PK136) or by MACS using EasySep™ Mouse CD8+ or CD4+ T Cell Isolation Kits (STEMCELL). 1–2×104 sorted cells were then incubated on 96-well u-bottom plates pre-coated with anti-CDƐ (145–2C11) antibody and cultured in complete RPMI containing 0.01–0.1μg IL-12-Fc or an equivalent volume of PBS. After 48 hours incubation, IFNγ was measured by CBA using Mouse IFNγ Flex Set (558296, BD) according to the manufacturer’s protocol. For human PBMCs, cells were sorted by MACS using EasySep™ Human CD8+ (17953, STEMCELL) or CD4+ (17952, STEMCELL) T Cell Isolation Kits according to manufacturer’s protocol. 1×104 sorted cells were then incubated on 96-well u-bottom plates in complete RPMI with ImmunoCult™ Human CD3/CD28 T Cell Activator (10971, STEMCELL), adding either 0.1μg of hu-IL-12-Fc or an equivalent volume of PBS. After 48 hours incubation, IFNγ was measured by R&D Human IFN-gamma DuoSet™ ELISA Kit (DY285B, Fisher) according to the manufacturer’s protocol.
Mouse ifn γ flex set
Manufactured by BD
Sourced in United Kingdom
The Mouse IFN-γ Flex Set is a multiplex assay kit designed for the quantitative measurement of mouse interferon-gamma (IFN-γ) in biological samples. The set provides the necessary reagents to perform the assay, including capture and detection antibodies, standards, and buffers.
Automatically generated - may contain errors
Lab products found in correlation
Mouse tnf flex set, by BD (2 mentions)
Easysep mouse cd8 or cd4 t cell isolation kits, by STEMCELL (1 mentions)
Immunocult human cd3 cd28 t cell activator, by STEMCELL (1 mentions)
Facsmelody, by BD (1 mentions)
Mouse il 17a flex set, by BD (1 mentions)
Mouse il 6 flex set, by BD (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Legendplex mouse inflammation panel, by BioLegend (1 mentions)
Mouse il 10 flex set, by BD (1 mentions)
4 protocols using mouse ifn γ flex set
1
Cytokine-Induced IFN-γ Production
2
Anti-tumor Effects of SZYY in Cells
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High glucose Dulbecco’s modified Eagle’s Medium (DMEM, GIBCO, PA, USA). Fetal bovine serum (FBS, EVERY GREEN, China). Penicillin/streptomycin (P/S, GIBCO, USA). AG490 (HY-12000, MCE). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China). Docetaxel (114977–28-5, aladdin, shanghai, china). IL-6 (Mouse IL-6 Flex Set, 558301, BD), IL-17A (Mouse IL-17A Flex Set, 560283, BD), TNF-α (Mouse TNF Flex Set, 558299, BD), IFN-γ (Mouse IFN-γ Flex Set, 558296, BD). DAPI staining reagent (G1012, servicebio, Wuhan, China). SZYY obtained from C. officinalis Planting Base in Taiping Town, Xixia County, Nanyang City, Henan Province, China.
3
Cytokine Profiling in Tumor Microenvironment
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Tumor interstitial fluids were obtained by manual cryopulverization and subsequent incubation with 1:2 tumor weight/volume of 2 × PBS for 3 h at 4°C under rotation. The LEGENDplex™ mouse inflammation panel (Biolegend) was used to determine cytokines levels in the tumor supernatants. To quantify protein levels in NK/PyMT cell co-culture supernatants, ELISA kit for PRF1 (Abbexa, Cambridge, UK, abx258736) as well as the mouse IFN-γ Flex Set (BD Bioscience, 558296) were utilized according to the manufacturer's instructions. Bead-based array samples were acquired by flow cytometry and analyzed using FlowJo V10.
4
Cytokine Quantification in Mouse Serum
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The contents of IL-10 (Mouse IL-10 Flex Set, 558300, BD), IFN-γ (Mouse IFN-γ Flex Set, 558296, BD), TNF (Mouse TNF Flex Set, 558299, BD), and IL-2 (Mouse IL-2 Flex Set, 558297, BD) in the serum of mice were detected using CBA according to the manufacturer’s protocols. Initially, the different concentrations of standard products were formulated according to the respective preparations, and a standard curve was plotted. Then, 50 μL of serum and 50 μL of microspheres were mixed and incubated at room temperature in the dark for 2 h. Then, 50 μL of PE antibody conjugated with TNF, IL-2, IL-1β, and IFN-γ was added and incubated for 1 h at room temperature in the dark. Further, 1 mL of washing solution was added and centrifuged, and the supernatant was gently discarded. The resulting suspension was resuspended in 400 μL of the buffer, and finally, the cytokine levels were measured by FCM, and the results were analyzed using the Diva (BD).
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