Splenocytes were suspended in complete media (RPMI 1640 with 10% FBS) at a concentration of 2 × 106 cells per mL. The cells were stimulated with or without 10 μg mL−1 of protein at 37 °C for 5 days. The supernatants were collected, and the levels of IFN-γ and IL-17A were determined by ELISA using mouse IFN-γ ELISA and IL-17A ELISA kits (Biolegend), respectively.
Mouse ifn γ elisa
Manufactured by BioLegend
Sourced in United States
The Mouse IFN-γ ELISA is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed to measure the concentration of mouse interferon-gamma (IFN-γ) in biological samples.
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Lab products found in correlation
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Cck 8 kit, by Dojindo Laboratories (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
X100 100ml, by Merck Group (1 mentions)
Human ifn γ, by BioLegend (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
8 protocols using mouse ifn γ elisa
1
Splenocyte Stimulation Assay for Cytokine Production
2
Quantifying Cytokine Levels in Serum
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Mouse IFN-γ ELISA (BioLegend, SanDiego, USA) and mouse IL-6 ELISA (eBioscience, SanDiego, USA) were applied according to the manufactures instructions to determine the amounts of these cytokines in serum.
3
Measuring Cytokine Levels in Stimulated Splenocytes
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The amounts of IFN-γ and IL-17A in the cell culture supernatants were determined based on a previously described method30 (link). Spleens were aseptically removed, and cells were suspended at a concentration of 2 × 106 cells/mL in complete media (RPMI 1640 with 10% FBS). The cells were stimulated with or without 10 μg/mL of IsdB, ClfA33-213, or IC protein at 37 °C for 5 days. The supernatants were collected, and the amounts of IFN-γ and IL-17A were determined by ELISA using mouse IFN-γ ELISA and IL-17A ELISA kits (Biolegend), respectively.
4
Splenocyte Proliferation and Cytokine Analysis
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Splenocyte proliferation assay was performed using CCK-8 kits (Dojindo, Japan). Splenocytes were suspended in complete media (RPMI 1640 with 10% FBS) at a concentration of 2.5 × 106 cells per mL. The cells were stimulated with or without 10 μg mL−1 of protein at 37 °C for 3 days. The results were expressed as the proliferation index (PI), which was calculated based on the following formula: PI = OD (450 nm) for stimulated cultures/OD (450 nm) for non-stimulated cultures.
The supernatants were collected for cytokine assay and the levels of IFN-γ, IL-4 and IL-17A were determined through ELISA using mouse IFN-γ ELISA, IL-4 ELISA and IL-17A ELISA kits (Biolegend, San Diego, CA, USA), respectively.
The supernatants were collected for cytokine assay and the levels of IFN-γ, IL-4 and IL-17A were determined through ELISA using mouse IFN-γ ELISA, IL-4 ELISA and IL-17A ELISA kits (Biolegend, San Diego, CA, USA), respectively.
5
Evaluating CAR T Cell Cytotoxicity
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Mock or anti-CEA CAR T cells were incubated with either MC38/GFP, MC38/CEA/GFP, E0771/GFP, or E0771/CEA/GFP cells at 0.1:1, 0.5:1, 1:1, 2:1, 5:1, or 10:1 ratio (Effectors:Targets, E:T) in RPMI 1640 medium, no phenol red (Gibco, 11835030) with 10% FBS, 2 mM L-glutamine, antibiotic-antimycotic solution, and β-mercaptoethanol on 96 wells (Eppendorf, 951040145) overnight at 37°C. For controls, only target cells were plated. For immunocytokine (ICK) studies, ICK (12 ng/mL; equivalent to 1 ng/mL IL2) was added to media along with T cells and target cells at the beginning of 24 hours co-culture. For positive controls, 20% Triton x-100 (Sigma-Aldrich, X100-100ML) in PBS was added to wells with only target cells and incubated for 30 minutes at 37°C. Supernatants were removed and collected for measurement of IFNγ by ELISA. For quantitative analysis of GFP, culture media were replaced with fresh 200 µL of RPMI 1640 medium without phenol red and GFP fluorescence was read on a CLAROstar instrument. Mouse IFNγ ELISA (BioLegends, 430806) was performed per manufacturer’s directions. Supernatants were collected from each well of in vitro killing assay plate and diluted 1:5 for IFNγ ELISA.
6
Mature moDC Cytokine and Surface Marker Analysis
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Mature moDC cell culture supernatants were tested for the levels of IL-12 (mouse IL-12 ELISA cat# 433607 and human IL-12 cat# 431704) and IFNγ (mouse IFNγ ELISA cat# 430804 and human IFNγ cat# 430104) using enzyme-linked immunosorbent assay (ELISA) kits (BioLegend). Mature DCs were stained immediately for flow cytometry. moDC differentiation and maturation was determined via labeling with fluorescence-conjugated antibodies specific for MHC class I (cat# 311403) and class II (cat# 361706), CD80 (cat# 305219), CD252 (cat# 326307), CD40 (cat# 334309), CD86 (cat# 305421), CD83 (cat# 305305), CCR7 (cat# 353203), CCR5 (cat# 313707), CCR6 (cat# 353415), CCR3 (cat# 310707) and CD205 (cat# 342203) (BioLegend). The expression of these cell surface markers was determined by flow cytometry and analyzed using FlowJo software (TreeStar).
7
Quantification of MCMV Infectivity
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In vivo infectivity was determined from homogenates of infected organs by plaque assay on MEF under conditions of centrifugal enhancement of infectivity. An infectious center assay was performed to determine the number of infected leukocytes in blood. Briefly, 4 days p.i., peripheral blood leukocytes (PBLs) were prepared from non-coagulated blood, counted, added to sub-confluent MEF monolayers in 12 well plates and overlayed with carboxymethyl cellulose. Three days later, the numbers of plaques were counted. For quantitation of viral DNA in PBLs, DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Then, viral genomes were quantitated by MCMV M55 (encoding gB)—specific qPCR normalized to cell numbers by pthrp specific qPCR [50 ]. Spleen cytokine levels were determined in organ homogenates using commercial ELISAs: mouse IFN-γ ELISA (Biolegend) and mouse MCP1 (CCL2) ELISA (Biolegend).
8
Splenocyte Activation and IFNγ Production
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