Determining the right incubation time is key to successful ELISpot and FluoroSpot experiments. This guide is designed to help you optimize incubation for accurate results, making your experimental process smoother and more reliable.
In our ELISpot and FluoroSpot kit protocols, you'll always find recommended incubation times for assessing each individual cytokine or immunoglobulin. It’s particularly crucial to optimize these times in FluoroSpot assays involving multiple analytes. A useful guideline is to base your incubation time on the analyte with the slowest secretion rate (i.e. the time it takes for the least rapidly produced analyte in the assay to reach detectable levels). This approach, however, might require adjusting cell numbers for specific analyte combinations. Such optimization ensures that rapidly secreted analytes do not lead to overlapping or confluent spots, which can compromise the accuracy of your results.
Different analytes are secreted at different rates or only after a certain amount of time. Finding that optimal window is key to successful FluoroSpot assays.
The risk of being unable to read confluent spots decreases significantly with the use of IRIS 2, thanks to our innovative RAWspot technology. This novel algorithm enhances spot detection by focusing on signal analysis rather than traditional image processing, ensuring more accurate and reliable readings.
Human analyte incubation times
| Analyte | Incubation time (hours) |
|---|---|
| EBI3 | 18-24 |
| GM-CSF | 18-24 |
| Granzyme B | 48 |
| IFN-α | 18-48 |
| IFN-γ | 12-48 |
| IgA | 16-24 |
| IgE | 16-24 |
| IgG | 16-24 |
| IgG1 | 16-24 |
| IgG2 | 16-24 |
| IgG2a | 16-24 |
| IgG2b | 16-24 |
| IgG2c | 16-24 |
| IgG3 | 16-24 |
| IgG4 | 16-24 |
| IgM | 16-24 |
| IL-10 | 18-48 |
| IL-12 (p70) | 18-48 |
| IL-12/-23 (p40) | 18-48 |
| IL-13 | 18-48 |
| IL-17A | 18-48 |
| IL-17A/F | 18-48 |
| IL-17F | 18-48 |
| IL-1α | 18-48 |
| IL-1β | 18-48 |
| IL-2 | 18-48 |
| IL-21 | 48 |
| IL-22 | 18-48 |
| IL-23 | 18-48 |
| IL-27 | 18-48 |
| IL-29 | 18-48 |
| IL-3 | 40-48 |
| IL-31 | 18-48 |
| IL-4 | 48 |
| IL-5 | 18-48 |
| IL-6 | 18-48 |
| IL-8 (CXCL8) | 18-48 |
| IP-10 (CXCL10) | 18-48 |
| Perforin | 72 |
| TNF-ɑ | 18-48 |
We hope you found this guide useful and that it can help guide you with optimizing your next FluoroSpot or ELISpot experiment! Check out all of our ELISpot and FluoroSpot kits or design your own FluoroSpot combination with FluoroSpot Flex.