The CTL ImmunoSpot® platform permits maximized scientifically-validated single cell FlouroSpot analysis. At the unprecedented resolution of up to 1 in 800,000, ImmunoSpot® assays measure effector molecule secretion by individual T cells that have been stimulated by an antigen.
ImmunoSpot® assays therefore provide information on the numbers of the antigen-specific T cells present in a test cell population, typically PBMC. The frequency of the specific T cells within the PBMC reflects the magnitude of T cell immunity. Spot sizes record the amount of cytokine produced by the individual cells. Spot sizes can be increased for polyfunctional T cells and for recently activated T cells and can be decreased in states of anergy or immune suppression. Measuring different molecules that T cells secrete provides information on the effector lineage of T cell immunity.
Several helpful online videos are available on working with FlouroSpot assays and PBMC. These videos are linked throughout the text and indicated by the symbol "⦿." You may also visit the ImmunoSpot® YouTube channel to view our complete library. Subscribe to our channel to be notified when new videos are posted.
How FlouroSpot Assays Work
Principle of the FluoroSpot assay: The FluoroSpot assay is a modification of the ELISPOT assay, which allows for visualization of plate-bound analyte produced by single cells (”spots”).
A. A FluoroSpot plate with a low auto-fluorescent PVDF membrane is coated with an analyte-specific capture antibody.
B. Freshly isolated, thawed, or cultured cells are plated together with the antigen of interest and incubated to allow for the activation of the antigen-specific T cells and the induction of analyte secretion.
C. The cells are washed away leaving the antibody-bound analyte in the well.
D. A detection antibody that is specific for a different determinant of the analyte is added – in this example this antibody is directly enzyme-coupled. The labeling of the detection antibody can also be done indirectly, via a streptavidin-biotin reaction.
E. A detection antibody that is specific for a different determinant of the analyte is added along with a fluorochrome. The use of different detection antibodies in conjunction with different fluorochromes permit multi-color detection of multiple analytes.
Principle of the Test
The assay can be done with freshly isolated PBMC, or with PBMC that have been frozen and thawed following optimized protocols. Along with the trial ImmunoSpot® Kit, CTL offers PBMC that have pre-defined reactivity to antigen along with the antigen (ePBMC® Reference Samples QC Set™). Such reference samples are indispensable for assay development, qualification, and validation.
Specifically designed 96-well PVDF plates are coated with a cytokine-specific monoclonal capture antibody (e.g., specific for IFN-γ). PBMC (or purified T cell subsets with APC) are pipetted into the pre-coated wells, and are cultured with the test antigen(s). Negative control wells contain either irrelevant antigen, or media alone. As positive control we recommend the use of CEF peptides to elicit CD8 cells, inactivated CMV virus for activation of CD4 cells, or PHA to stimulate both T cell subsets. The ePBMC® Reference Samples QC Set™ is ideally suited as reference standards for the assay’s performance. The activated T cells secrete molecules that are captured by the membrane-bound antibody.
For the best results in an FlouroSpot assay, the cells should be cultured with antigen for a time period that induces maximal cytokine (or other analyte) production: 24 hours are optimal for measuring IFN-γ secretion by T cells. After the activation culture, the cells are discarded (or can be transferred for further characterization/ propagation), and a labeled cytokine-specific detection antibody is added to the plate. Subsequently, the plate-bound detection antibody is visualized via an enzymatic reaction or fluorescence. ImmunoSpot® assays are optimized to detect and quantify in each color precipitation (“spot”) the footprint of a single cell’s secretory activity. Spot numbers therefore denote the accurate frequency of the antigen-specific T cells among the plated cells; spot sizes and morphology provide additional information on the magnitude and kinetics of the cells’ secretory activity.
Day 0 (Sterile conditions)
- Prepare Capture Solution by diluting the Capture Antibody according to your specific protocol.
- In a FluoroSpot assay, the membrane of a low auto-fluoroescent PVDF membrane should be prewetted with 70% ethanol for 30sec and washed with 150μl of PBS three times before adding 80μl of the Capture Solution into each well.
- ⦿ Incubate plate overnight at 4°C in a humidified chamber.
Day 1 (Sterile conditions)
- Prepare CTL-Test™ Medium by adding 1% fresh L-glutamine.
- Prepare antigen/mitogen solutions at 2X final concentration in CTL-Test™ Medium.
- Decant plate with coating antibody from Day 0 and wash one time with 150μl PBS.
- Plate antigen/mitogen solutions,100µl/well.
- After ⦿ thawing PBMC or ⦿ isolating white blood cells with density gradient, adjust PBMC to desired concentration in CTL-Test™ Medium, e.g.: 3 million/ml corresponding to 300,000 cells/well (however, cell numbers can be adjusted according to expected spot counts since 100,000-800,000 cells/well will provide linear results). While processing PBMC and until plating, ⦿ keep cells at 37°C in humidified incubator, 5-9% CO2.
- ⦿ Plate PBMC, 100µl/well using large orifice tips. Once completed, gently tap the sides of the plate and immediately place into a 37°C humidified incubator, 5-9% CO2.
- Incubate for 24-72 hours depending on your cytokine. (See your specific cytokine protocol for optimization.) Do not stack plates. Avoid shaking plates by carefully opening and shutting incubator door. Do not touch plates during incubation.
- Prepare Wash Solutions for the day: PBS, distilled water and Tween-PBS.
- Prepare Detection Solution by diluting Detection Antibody according to your specific protocol.
- Wash plate two times with PBS and then two times with 0.05% Tween-PBS, 200µl/well each time.
- Add 80µl/well Detection Solution. Incubate at RT, 2h.
- Prepare Tertiary Solution by diluting the Tertiary Antibody according to your specific protocol.
- Wash plate three times with 0.05% Tween-PBS, 200µl/well.
- Add 80µl/well of Tertiary Solution. Incubate at RT, 1h.
- Decant and wash plate three times with distilled water, 200µl/well. (CTL has seen optimal results when the last water wash is filtered through with a vacuum manifold to eliminate any unbound tertiary.)
- Air dry plate for 2 hours face-down in running hood or on paper towels for 24 hours on bench top.
- Scan and count plate. (Discount scanning analysis services are offered with the purchase of ImmunoSpot® Kit.)
- For in vitro and research use only – Not intended for use in diagnostic procedures.
- Blood samples, PBMC, or cell isolates should be treated as potentially infectious biohazard. Handle everything that comes in contact with specimen as potential biohazard, level II.
- Wear disposable gloves and personal protection throughout the entire procedure and thoroughly wash hands after handling the test reagents.
- Wipe any spills immediately with a laboratory-approved disinfectant, such as 10% bleach followed by 70% ethanol.
- All test specimens and materials used in the procedure must be disposed of as biohazardous waste.
- It is important to quick-spin vials before use to ensure content volumes.
- ⦿ To maximize the use of each plate, an adhesive plate-sealing sheet has been included that can be adhered to the top of the plate to cover and protect unused wells that are intended to be used in subsequent assays. If such is intended, use your fingers & thumbs to firmly adhere the sheet to the plate and use a razor blade to cut the sheet to expose only the necessary wells.
- We highly recommend the use of CTL CTL Serum-free Media for freezing, washing, and testing PBMC. Even brief exposure to a mitogenic serum can cause high background while other sera can have suppressive effects. CTL also recommends using the CTL-LDC™ Kit for accurate live/dead cell counts.
- Deviations from specified temperatures, timing requirements, number of washing steps, and specified reagent preparation volumes may alter the performance of the assay.
- Plates can be washed manually or by a suitable automated plate washer with adjusted pin length and flow rate so membranes and spots are not damaged.
- To avoid damage to the PVDF membrane in the wells, avoid touching the membrane with pipette tips or with the plate washer. The PVDF membrane is permeable and protected by an underdrain. Avoid direct contact between the well bottom and wet surfaces, including paper towels or any other materials that can absorb liquid.
- While processing plates, the PVDF membrane at the bottom of the wells must remain wet.
- When underdrain and gloves are wet, the underdrain may be slippery and difficult to remove. Wipe gloves and underdrain with paper towel before removing.
- ⦿ After completion of the experiment, do not dry the ELISPOT assay plates at temperatures exceeding 37°C as this may cause the membrane to crack.
- Spots may not be readily visible while the membrane is still wet. Scan and count plates with a CTL ImmunoSpot® Analyzer only after membranes have completely dried.
- Higher background appearing in the control wells can be potentially overcome by the following steps:
- When working with pre-cultured cells, wash the cells thoroughly in CTL-Wash™ prior to the experiment in order to avoid carryover of cytokines and other substances; use CTL-Test™ for testing PBMC.
- The SmartCount™ module of the ImmunoSpot® Software automatically recognizes spots over high background or uneven background and will correct background deviations. The Autogating™ module will help discern between T cell-derived and background spots. The CTL technical support team will gladly assist you with using the ImmunoSpot® Software for the analysis of complicated test results.
- Data analysis. The CTL ImmunoSpot® Analyzers along with the ImmunoSpot® Software have advanced features that permit automated, objective recognition of spots, gating and counting.
- The CTL team will gladly assist you with data analysis and troubleshooting, as well as customizing FlouroSpot assays to suit your needs. Please contact us via email at email@example.com..