Human IFN-gamma/Granzyme B Dual-Color FluoroSpot Kit Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
ELISpot kits are highly sensitive, microplate-based assays for the fluorescent detection of cytokine secreting cells. This kit is designed for the simultaneous detection and enumeration of Human IFN-gamma/Granzyme B.
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.
- 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
- 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
- 35% ethanol or methanol
- Block Buffer (1% BSA and 5% sucrose in PBS)
- ELISpot reader capable of detecting fluorescence or a conventional epifluorescence microscope
Preparation and Storage
Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
Do not use reagents from this kit with components from other R&D Systems ELISpot or ELISA kits and/or components manufactured by other vendors.
Do not remove the flexible plastic underdrain on the bottom of the microplate before or during incubation and development since it may damage the PVDF membrane filters. The underdrain cover may be removed only after completing the incubation with chromogen.
Wash Buffer: If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. To prepare the Wash Buffer, add 50 mL of Wash Buffer Concentrate to 450 mL of deionized water and mix well.
Positive Controls: Reconstitute as recommended in the product datasheet using the same culture medium that is used to incubate cells.
Capture Antibody Mixture: Tap or vortex each vial to release the reagent collected in the cap. Dilute as recommended in the product datasheet and mix well. For optimal performance, prepare the Capture Antibody Mixture immediately before use.
Detection Antibody Mixture: Tap or vortex each vial to release the reagent collected in the cap. Dilute as recommended in the product datasheet and mix well. For optimal performance, prepare the Detection Antibody Mixture immediately before use.
Streptavidin-NL557: Tap or vortex the vial to release reagent collected in the cap. Dilute as recommended in the product datasheet and mix well. For optimal performance, prepare the Streptavidin-NL557 immediately before use.
The types of effector and responder cells used, method of cell separation, mode of stimulation, and length of incubation are to be determined by each investigator. R&D Systems cell selection products are suitable for the purification of effector and responder cells.
Bring all reagents to room temperature as needed, except the Antibody Concentrates and Dilution Buffer 1, which should remain at 2 °C – 8 °C. All samples and controls should be assayed at least in duplicate. An Assay Record Template is provided in the product datasheet to record the location of the controls and samples assayed.
- Prepare the membranes by adding 15 μL of 35% alcohol to each well. Incubate for 1 minute.
- Aspirate each well and wash, repeating the process four times for a total of five washes.
Wash by filling each well with deionized or distilled water (250-300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining liquid by aspirating or decanting. Invert the plate and blot it against clean paper towels.
Note: Adjust the height of the manifold dispenser or autowasher to prevent damage to the membranes.
- Add 100 μL of diluted Capture Antibody Mixture into each well, and incubate overnight at 2-8 °C.
- Aspirate each well and wash with 1X PBS (as in step 2), repeating the process three times for a total of four washes.
- Fill each well of the microplate with 200 μL Block Buffer, and incubate the microplate for approximately 90 minutes at room temperature.
- Aspirate the Block Buffer, and fill each well of the microplate with 200 μL of sterile culture media. Incubate for approximately 20 minutes at room temperature.
- When the cells are ready to be plated, aspirate the culture media from the wells. Immediately add 100 μL of the appropriate cells or controls to each well. Refer to the Technical Hints section for appropriate controls.
- Incubate the cells in a humidified 37 °C CO2 incubator. Optimal incubation time for each stimulus should be determined by the investigator. Do not disturb the cells during the incubation period.
- Aspirate each well and wash with Wash Buffer (as in step 2), repeating the process three times for a total of four washes.
- Add 100 μL of diluted Biotin-conjugated Detection Antibody to each well, and incubate overnight at 2-8 °C.
- Repeat the wash described in step 9.
- Add 100 μL of diluted A488-conjugated Detection Antibody + Streptavidin-NL557 Mixture to each well, and incubate for 2 hours at room temperature. Protect from light.
- Repeat the wash described in step 9 using 1X PBS instead of Wash Buffer.
- Add 100 μL of NorthernLights Fluorescence Enhancer Solution to each well, and incubate for 15 minutes at room temperature. Protect from light.
- Aspirate the NorthernLights Fluorescence Enhancer Solution. Remove the under drain from the back of the microplate, and rinse with deionized or distilled water. Do not rinse the wells. Let the microplate dry completely before analyzing. Protect from light until analysis.
CALCULATION OF RESULTS
The developed microplate can be analyzed by counting spots using either an ELISpot reader capable of detecting green and red fluorescent spots or a conventional epifluorescence microscope. Specific spots are round and have a dark center with slightly fuzzy edges. Quantification of results can be done, for example, by calculating the number of spot forming cells (SFC) per number of cells added to the well.
Please see the reproducibility data provided in the product datasheet.
TECHNICAL HINTS AND LIMITATIONS
- To minimize edge effect, place the microplate (bottom down) onto a piece of aluminum foil (about 4 x 6 inches). Add cells, cover the microplate with the lid, and shape the foil around the edges of the microplate. The foil may be left on the microplate until after the NorthernLights Fluorescence Enhancer has been aspirated.
- Do not remove the flexible plastic under drain on the bottom of the microplate before or during incubation and development. It may damage the PVDF membrane filters. The under drain cover may be removed only after completing the incubation with NorthernLights Fluorescence Enhancer.
- To avoid damage to the membrane, do not touch PVDF membrane filters with pipette tips when pipetting cells and reagents.
- Upon completing the experiment, do not dry the microplate at a temperature above 37 °C. It may cause the PVDF membrane filters to crack.
- The 96-well microplate provided in this kit is not sterile. Due to the short incubation period and the presence of antibiotics in the culture media, microbial contamination has not been shown to be an issue with this FluoroSpot procedure.
- This kit is designed for single use only. The layout of the assay should be carefully planned to maximize the use of the provided microplate and reagents.
- The controls listed are recommended for each FluoroSpot experiment:
- Positive Control - Use recombinant human IFN-gamma and recombinant human Granzyme B in separate wells.
- Unstimulated/Negative Control - Use the same number of unstimulated cells as stimulated cells.
- Background Control - Use sterile culture media.
- Detection Antibody Control - Substitute phosphate buffered saline for Detection Antibody.
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