Why resting cells is essential for accurate ELISpot and FluoroSpot

When it comes to ELISpot and FluoroSpot experiments, every detail counts. One essential step that can significantly impact the accuracy of your ELISpot or FluoroSpot experiment is the process of resting cells before analysis. Let’s dive into why this step is crucial and how to do it properly.

Why rest your cells?

In ELISpot and FluoroSpot assays, you measure the secretion of specific analytes, such as cytokines or antibodies, by individual immune cells. These assays are incredibly sensitive, capable of detecting even a single cell that secretes the analyte of interest. Cells can spontaneously secrete high levels of these analytes depending on in vivo stimulation or agitation during isolation, leading to skewed results*. Resting cells before ELISpot or FluoroSpot analysis helps ensure that the cells are in their natural, unstimulated state, providing a more accurate representation of their normal analyte secretion levels. Another important and critical reason for resting your cells is to remove cell debris, which leads to a cleaner assay.

How to properly rest your cells

• Isolate your cells: Start by isolating the cells of interest, whether they are PBMCs or enriched T cells, B cells, or other immune cells. Check out our guide on how we isolate immune cells in-house, normally leading to >95% viability.
• Adjust the concentration: After isolating and washing the cells, measure viability and adjust the concentration to an appropriate level for resting. Typically, this involves resuspending the cells in a cell culture medium at a concentration of around 1-2 million cells per ml.
  • We recommend resting cells in sterile 50 ml or 15 ml conical tubes with a  slightly loosened cap to allow for gas exchange when placing the tubes in the incubation. These tubes provide the necessary high-density culture conditions for optimal cell viability.
• Rest the cells: Incubate the cells in a sterile incubator at 37 °C with 5% CO₂. Allow the cells to rest for at least one hour or even overnight. The optimal resting time can vary depending on the cell type and the specifics of your assay and may require some optimization.

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• After the incubation period, cells should have returned to a resting state.

• Gently resuspend the rested cells and allow any clumped debris to fall to the bottom of the tube. While avoiding the clump, transfer the cell suspension to a new 50 ml tube for washing steps.

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• Wash the cells by centrifugation and pour off the supernatant. Resuspend in fresh media and repeat one more wash.

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• Check viability once more and adjust cell numbers to an appropriate concentration for the ELISpot or FluoroSpot assay.

Remember, the devil is in the details! Taking the time to properly rest your cells can make all the difference in obtaining accurate and reliable data. Feel free to reach out to our team if you have any questions!

*Unspecific stimulation can occur due to improper procedures in animal facilities. Contaminations in these facilities can introduce significant variability and bias in experimental results, jeopardizing the validity and reproducibility of scientific research. Regular testing and stringent hygiene standards are crucial to maintaining a controlled environment, ensuring the accuracy and reliability of data, and upholding the integrity of scientific investigations.

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