Blood samples were obtained from healthy donors. Spleens were obtained from BALB/c or C57BL/6 mice housed with ethical approval from the Cantonal Veterinary Office of Zürich, Switzerland (license ZH215/17). Spleens were dilacerated and splenocytes were resuspended in 10 mL of complete medium. For both human and mouse cells, mononuclear cells were isolated using the Ficoll-Paque™ Plus (GE Healthcare Life Sciences, Marlborough, MA, USA) method. A total number of one million cells per well were plated on 96-well plates and incubated overnight with appropriate RNA carrier containing 200 ng of RNA per well (200 μL cultures). Protamine RNA particles were prepared as described previously [14 ,15 (link)]. The next day, supernatants were taken, and IFNα concentrations were measured via ELISA by following the manufacturer’s protocol (Human IFNα pan ELISA development kit, MABTECH, ELISA MAX Standard Set Mouse IFNα, Biolegend, San Diego, CA, USA). The absorbance was measured at 450 nm with an ELISA reader (GloMax Discover and Explorer Detection System equipment, Promega, Madison, WI, USA) and cytokine concentrations were calculated according to a standard curve.
Glomax discover and explorer detection system equipment
Manufactured by Promega
Sourced in United States
The GloMax Discover and Explorer Detection System is a versatile laboratory equipment designed for detecting and measuring various luminescent and fluorescent signals. It offers high-sensitivity detection capabilities for a wide range of assays and applications. The core function of this system is to provide accurate and reliable data acquisition and analysis for researchers and scientists working in diverse fields.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (4 mentions)
Elisa max standard set mouse il 2, by BioLegend (2 mentions)
Ifn α, by Mabtech (1 mentions)
Human tnfα, by BioLegend (1 mentions)
Human ifn α, by BioLegend (1 mentions)
Tnf α, by BioLegend (1 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Merck Group (1 mentions)
5 protocols using glomax discover and explorer detection system equipment
1
IFNα Production in Human and Mouse Cells
2
Protamine-RNA Immunomodulatory Assay
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Blood was obtained from healthy donors, from which mononuclear cells were isolated using Ficoll- Paque™ Plus (GE Healthcare Life Sciences) method. A total number of one million cells per well were plated on 96-well plates and incubated overnight with appropriate RNA carrier containing 500 ng of RNA per well (200 μl cultures). Protamine-RNA particles were prepared as described previously.16 ,17 (link) The next day supernatants were taken, and IFNα and TNFα concentrations were measured via ELISA by following the manufacturer’s protocol (Human IFNα; Mabtech, human TNFα; BioLegend). In mice, serum was taken 4 h after injection of Protamine/RNA particles, and measured for IFNα (MABTECH) and TNFα (BioLegend). The absorbance was measured at 450 nm with an ELISA reader (GloMax Discover and Explorer Detection System equipment, Promega) and cytokine concentrations were calculated according to a standard curve.
3
Ovalbumin mRNA Immune Response Assay
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C57BL/6-Tg(TcraTcrb)1100Mjb mice, also referred to as OT1 mice, were a generous gift from Pål Johansen (University Hospital Zurich). Spleen mononuclear cells from OT1 mice were isolated using the Ficoll-Paque™ Plus (GE Healthcare Life Sciences, Marlborough, MA) method. A total number of 150,000 cells per well (in 200 μL culture) were plated on 96-well plates and incubated for 24 h with 200 ng (1 µg /mL) of mRNA coding for Ovalbumin, formulated either in Lipofectamine MessengerMax (ThermoFisher) or in Protamine-RNA particles. After 24 h, supernatants were taken, and Interleukin-2 (IL-2) concentration was measured via ELISA, as per the manufacturer’s protocol (ELISA MAX Standard Set Mouse IL-2, Biolegend, San Diego, CA, USA). The absorbance was measured at 450 nm with an ELISA plate reader (GloMax Discover and Explorer Detection System equipment, Promega, Madison, WI, USA).
4
Cell Cytotoxicity Assay for Chemotherapeutics
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Cells (5000 cells per well) were seeded in 300 μl of complete medium in a 48-well plate. One day later, supernatants were replaced by 200 μl of complete medium. Increasing concentrations of free 5FU or Protamine/RNA nanoparticles incorporating chemotherapeutic purine analogues Ribavirin, Nebularine or 5FU were added to the cells. Protamine/RNA was used as a controls. After three days cell cytotoxicity is measured using the Cell Counting Kit-8 (CCK-8, Sigma). Cells were incubated with CCK-8 solution and incubate at 37°C for 6 h. Absorbance is measured at OD 450 nm using the GloMax Discover and Explorer Detection System equipment (Promega).
5
Quantifying T Cell IL-2 Production
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C57BL/6-Tg(TcraTcrb)1100Mjb (22) mice, also referred to as OT1 mice, were a generous gift from Pål Johansen (University Hospital Zurich). Spleen mononuclear cells from OT1 mice were isolated using the Ficoll-Paque™ Plus (GE Healthcare Life Sciences, Marlborough, MA) method. A total number of 200,000 cells per well (200 μL culture) were plated on 96-well plates and incubated overnight with appropriate mRNA carrier containing 200 ng of immunostimulating mRNA per well. The next day, supernatants were taken, and interleukin-2 (IL-2) concentration was measured via ELISA, as per the manufacturer’s protocol (ELISA MAX Standard Set Mouse IL-2, Biolegend, San Diego, CA, USA). The absorbance was measured at 450 nm with an ELISA plate reader (GloMax Discover and Explorer Detection System equipment, Promega, Madison, WI, USA).
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