APPLICATIONS IN ACTION CELLS AND BEADS – BETTER TOGETHER Simultaneous Analysis of Secreted Il-2 and Jurkat Cells SUMMARY The iQue Screener can analyze mixtures of cells and beads. •QBeads™ and Jurkat cells can be analyzed simultaneously on the iQue Screener •Jurkat cells secrete IL-2 within 20 hours of PHA treatment •Simultaneous cells and beads analysis on the iQue adds contextual value •Analyzing both cells and QBeads in the same sample are easily distinguished based on their scatter characteristics, providing better biological content P P B PROBLEM Jurkat cells are an immortalized human T-cell line that is useful as a surrogate for T-cells in many biological studies including T-cell signaling, cancer therapies, and viral infections. Measuring the production and secretion of IL-2 is a critical component in many biological applications. For example, IL-2 is a key part of the T-cells response to bacterial infection. IL-2 secretion activates T-cells to proliferate and differentiate. It is required in the process of discriminating self from non-self, and thus is relevant in all auto-immune disorders. Many immunosuppressant drugs inhibit the secretion of IL-2 by activated T-cells. IL-2 has also been demonstrated to be involved in the induction of pruritus. Clearly the ability to screen for both secreted IL-2 and T-cells simultaneously has merit. RESULTS The QBeads™ PlexScreen kits can detect and quantify any of 50 human secreted proteins, including IL-2 in cell culture media or serum. All of the QBeads PlexScreen products have been validated in one wash and no wash protocols; the no wash protocol was used in all experiments noted, including the standard curve in Figure 1. On the iQue® Screener system, cells and QBeads in the same sample are easily distinguished based on their scatter characteristics (Figure 1). This separation of cells and beads and the ability to discriminate them is maintained when other cell types or even complex cell mixtures such as PBMCs are utilized. Figure 1. Background graphics. A. QBeads are separated from the Jurkat cells on the iQue Screener by their light scattering qualities. B. A standard curve of human IL-2 used QBeads and a no wash assay protocol. Data is presented as average + std dev. of triplicate values. APPLICATIONS IN ACTION | CELLS AND BEADS – BETTER TOGETHER Simultaneous analysis of beads and cells does not affect data quality. To test for any impact on data quality from analyzing samples that contain both QBeads and cells, a dose response of phytohemagglutinin (PHA) was performed. PHA is a known mitogen that is a surrogate for bacteria in activating T-cells. Following PHA treatment, the assay was divided; one half was centrifuged to remove the Jurkat cells. The other half was not centrifuged and so contained the cells. QBeads were then added to both and analyzed on the iQue. There is no significant difference in IL-2 detection between supernatant plus QBeads and cells plus QBeads run on the iQue Screener (Figure 2). In fact, while there is no detriment to the combined assay for IL-2 secretion, there are benefits to analyzing both cells and beads simultaneously. One such benefit is better biological content. Figure 2. Simultaneous analysis of cells and beads. Jurkat cells were treated with PHA for 20 hours and secreted IL-2 was analyzed alone or in the presence of Jurkat cells. The assay was read on an iQue Screener, following the no wash protocol. By analyzing both cells and beads simultaneously, one is able to make correlations between the various responses and gain contextual value to the data. Figure 3 shows another PHA dose response, this time with phorbol 12-myristate 13-acetate (PMA) co-stimulation. The highest doses of PHA used in this experiment also caused cell death that was dose dependent, even while the IL-2 production peaked. For example, the amount of IL-2 produced per cell increased even as the number of live cells substantially decreased. This type of insight can be valuable when triaging lead compounds, or optimizing a treatment or growth conditions. THE INTELLICYT SOLUTION • Cells and QBeads in the same sample are easily distinguished based on their scatter characteristics - This separation of cells and beads and the ability to discriminate them is maintained when other cell types or even complex cell mixtures such as PBMCs are utilized. • Achieve better biological data – Distinguish live from dead cells in real time while simultaneously analyzing secretions per cell. • Flexible – Analyzes 96, 384 and 1536 well plates in any plex size up to 30 Visit www.intellicyt.com to learn how iQue can improve your screens! +1•505•345•9075 • Figure 3. IL-2 secretion and cell counts from co-stimulated Jurkat cells. A dose response of PHA on Jurkat cells that were co-stimulated with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA). Secreted IL-2 was measured with IL-2 QBeads PlexScreen kit following the no wash protocol. Cells were simultaneously counted in the same samples on an iQue Screener. Production of IL-2 on a per cell basis peaked at the 4000 ng/ ml of PHA treatment. Cell viability had diminished to less than 20% At the same dose. www.intellicyt.com Doc Number 11602.A ©2014 IntelliCyt Corporation. All Rights Reserved. The trademarks used herein are the property of IntelliCyt Corporation or their respective owners. FOR RESEARCH USE ONLY. NOT FOR IN VITRO DIAGNOSTIC USE.