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Flow cytometry is a powerful tool for analysis of cells and cell-based assays in basic research and for drug discovery and development. The applications of this technology are many and varied, ranging from simple cell counts and viability assays, to complex intracellular staining. But it is the ability to analyze large numbers of post transcriptional v changes, which have made flow cytometry indispensable for modern cell biology laboratories. Despite first appearing in the 1960s, and the countless advances since then, flow cytometry still has a reputation of being a difficult technique requiring dedicated experts with in-depth knowledge to perform successfully. Sartorius’ iQue® advanced flow cytometry platform has been developed to overcome this, putting this powerful technique back into the reach of every lab.
This case study addresses the demands of modern drug discovery and developmental workflow, exploring the below four key pillars of experimental design, to demonstrate the benefits that a comprehensive solution of accelerating cell based assay workflows will bring.
1. Reproducibility 2. Reliability 3. Optimized sample usage 4. Multiplexing
The iQue® 3 is an advanced high throughput flow cytometry solution aimed to speed up entire workflow. The iQue® platform offers a novel screening solution that streamlines the process from experimental setup and acquisition to analysis and results.
Designed specifically with drug discovery and development in mind, it requires minimal assay optimization thanks to an array of standardized protocols and reagents, and the system’s dedicated iQue Forecyt® software generates data such as IC50/EC50 curves, heatmaps, and graph plot analyses in real time. Rapid, accurate data generation is critical to project progression, so the iQue® was designed to provide greater biological insights from fewer experiments using less material. It can simultaneously measure cell-specific parameters—such as phenotypic changes and proliferation rates—and quantify secreted factors in a single assay, using volumes as low as 5 µL per well.
Reproducibility The iQue® has been engineered to produce robust data across replicates, showing good intra- and inter-day correlation between assay plates (Figure 1A and 1B). Wells containing a known quantity of reagent were used to test reproducibility over different plate replicates and days of testing. Four mouse antibody isotypes, IgG1, IgG2a, IgG2b and IgG3 were mixed in a wide range of concentrations and measured across 24 replicate wells.
Three plates were run on the same day (Figure 1A) or a single plate was measured on separate days (Figure 1B) using the iQue® Mouse IgG Type and Titer Assay Kit. Variability was analyzed across all samples, achieving a correlation R2 > 0.99, with linear regression curve values of ~ 1, showing outstanding assay reproducibility for both intra- and inter-day testing.
Optimized Sample Usage The sequential sampling and miniaturization capabilities of the iQue® allow multiple parameters to be evaluated from a single well over time, conserving valuable samples and reagents, and reducing experimental variability. To demonstrate this, the iQue® Multiplex Human Antibody Internalization Kit—an antibody internalization assay— was run for 20 hours with repeated evaluation of multiple parameters, including cell viability. Ramos cells were plated at 1×106 cells/mL in 384-well plates containing a two-fold serial dilution of antibodies—starting at 1 mg/mL—and sequential measurements were performed for each well at various time points. Heatmaps (Figure 4A) and graphs (Figure 4B) present data analysis for the degree of antibody internalization at every sampled time point, demonstrating the importance of evaluating multiple time points for detection of antibodies with slower internalization rates, as exemplified here by CD20. Repeated sampling with the iQue® is also ideal for tracking dynamic biological changes over time, such as quantification of cytokine secretion following activation of cells.
Reliability The iQue® is able to provide users with consistent data across different individual instruments or operators, offering dependable operation and remarkable consistency between runs using a fixed photomultiplier tube (PMT) voltage.
Multiplexing The last pillar, and yet of increasing demand among drug discovery labs, is the iQue®’s ability to perform multiparametric measurements to generate more biologically-relevant data. With validated, ready-to-use multiplexed assay kits, the iQue® is ideally placed to handle several plates and provide multiple readouts, using no wash | single wash assays and novel data reduction tools to deliver accurate and precise results while relieving bottlenecks in the laboratory workflow. The iQue® can simultaneously measure changes in cell phenotype, proliferation, and apoptosis—in combination with secreted protein release (e.g., cytokines), bringing a new meaning to high-content analyses.
The examples presented in this case study demonstrate the suitability of the iQue® as a reliable platform for high content, high-throughput cellular analyses. The system has been developed with drug discovery and development labs in mind, where quick, reproducible, and multiplexed screening platforms are in increasing demand. In conclusion, whether you are discovering a new therapeutic antibody, developing a specific checkpoint inhibitor or evaluating CAR T cell function, driving your research forward hinges on the ability to rapidly evaluate more samples in a biologically relevant, reproducible, and cost effective way. The iQue® 3 is the most advanced flow cytometry platform with a focus on speed from setup, to acquisition, and analysis. It is able to get you from samples to actionable results in record time. The iQue® 3 combines a patented sampling method, which allows for the fastest sample acquisition in the industry and has the ability to handle 96, 384, or 1536-well plates, and enables continuous plate loading through connection with any automation system. It’s simple, scalable, multi-user environment featuring walkaway automation, comprehensive analysis and visualization tools that are not available in traditional flow cytometers.
For more information: Application case study with Flow Cytometry Solutions
