Human IFN-gamma/Granzyme B Dual-Color FluoroSpot Kit

Capture antibodies specific for Human IFN-gamma/Granzyme B are coated on a 96-well PVDF-backed microplate. Appropriately stimulated cells are added to the microplate which is then placed in a humidified 37 °C CO₂ incubator for the desired period of time. During this incubation period, the immobilized capture antibodies in the immediate vicinity of the secreting cells bind secreted IFN-gamma/Granzyme B. At the desired time point, one analyte is detected using a biotinylated detection antibody and NorthernLights™ (NL) 557-conjugated to Streptavidin (red flurophore). The second analyte is detected using a NL493-conjugated detection antibody (green fluorophore). Following incubation with the NorthernLights Fluorescence Enhancer, spots form to indicate the presence of IFN-gamma/Granzyme B secreting cells. Each spot represents an individual secreting cell. The spots can be counted with an ELISpot reader system or using an epifluorescence microscope.

Product Features for Human IFN-gamma/Granzyme B Dual-Color FluoroSpot Kit

• Detect and quantitate individual cells secreting IFN-gamma/Granzyme B
• High sensitivity - ELISpot assays can measure responses with frequencies well below 1 in 100,000 cells
• No in vitro expansion of cells required
• ELISpot assays use only a small number of primary cells

Kit Contents for Human IFN-gamma/Granzyme B Dual-Color FluoroSpot Kit

• PVDF-backed Microplate
• Capture Antibody Concentrates
• Biotin-conjugated Detection Antibody
• Streptavidin-conjugated to NL557
• A488-conjugated Detection Antibody
• Dilution Buffers
• Wash Buffer Concentrate
• NorthernLights Fluorescence Enhancer
• Human IFN-gamma/Granzyme B Positive Controls

Other Reagents Required

• Pipettes and pipette tips
• Deionized or distilled water
• Squirt bottle, manifold dispenser, or automated microplate washer
• 500 mL graduated cylinder
• 37 °C CO₂ incubator
• Sterile culture media
• PBS
• 35% ethanol or methanol
• Block Buffer (1% BSA and 5% sucrose in PBS)
• ELISpot reader capable of detecting fluorescence or a conventional epifluorescence microscope