Search

A need for more robust compensation techniques comes with continued growth in the study of immunotherapy and immunology by flow cytometry. Compensation is a method used to address the measurement of a fluorophore in multiple detectors (referred to as spillover) by mathematically correcting signal overlap between emission spectra of different fluorochromes to determine the true level of staining/positive target signal. The signal spillover can impact data quality and becomes very important with different levels of marker expressions. This measurement can be achieved by use of cells, or more typically by use of compensation beads to save precious biological samples. Current compensation beads have been shown to be problematic and can lead to over- or under-compensation, resulting in less or more true positive signal, respectively. Additionally, current compensation beads are incompatible with far red violet and ultraviolet dyes and may not be ideal for dim/low expressing markers. These studies address compensation by comparing various compensation products to biological samples on multiple immunophenotyping panels of either human or murine origin. These panels compare the use of a wide range of fluorophore conjugates, including the use of violet and ultraviolet excitable dyes, low expressing targets, and intracellular targets to determine compensation accuracy. The results demonstrate that a precise compensation matrix can be obtained with a newly improved commercial bead that is comparable to a cell sample. This data validates that precious cell samples may not be needed to be used in place of commercial bead products to obtain accurate compensation and quality data regardless of a panel’s design elements.

Cellular responses to drug treatment can be varied and heterogeneous. Often, a multiparametric approach is necessary to identify cellular pathways that are responding or affected. To maximize the number of parameters analysed per sample, we aimed to combine several functional sensors into one assay. In addition, we set out to streamline the workflow focusing on live cell responses. To this end, we designed a flow cytometry-based detection system centered on a single incubation condition for sensors that have minimal spectral overlap. Analysing Jurkat cells treated with camptothecin, a topoisomerase inhibitor that triggers cellular apoptosis, we were able to simultaneously detect the loss of cells in G2/M phase using the stoichiometric DNA dye, Hoechst 33342; an increase in apoptotic cells with a sensor for activated caspase 3/7 (CellEvent Caspase-3/7 Green); the loss of mitochondrial membrane potential with tetramethylrhodamine, methyl ester (TMRM); and an increase in dead cells as well as cells in late apoptotic stage, using a SYTOX viability dye. We also tested another four-sensor combination that focused on detecting cellular stress. Upon prolonged camptothecin induction, we were able to detect an increase in mitochondrial stress with MitoSox, a sensor that detects mitochondrial oxidative stress; an increase in cellular stress with a CellROX reagent specific for reactive oxygen species. There was also a loss in mitochondrial membrane potential as well as a greater loss of cellular viability. In summary, using a model of cellular response to drug treatment, we performed multiparametric cell status analysis with a combination of cellular detection reagents on a flow cytometer.

High throughput screening (HTS) is extremely effective method for allowing researchers to identify putative compounds of interest which play a role in their area of cellular research. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry not only allows a researcher to study drug effectiveness towards different cell types but also can be used to analyze protein-protein interactions, metabolic activities, as well as DNA/mRNA content in a single or multiplexed assay format. In this study, we screened the MicroSource Discovery System Killer Plate compound library and compared HTS using a plate reader with flow cytometer multiplexing. Jurkat and Ramos cells, T and B lymphocyte cell types respectively, were used as cell models and cultured under standard conditions and screened either at hypoxic (1%) or hyperoxic (19%) oxygen levels at varied lengths of time (24, 48, or 72 hrs). To assess compound veracity, post-screening analysis was implemented to establish EC50s of “hits” from the compound library. Membrane integrity and metabolic activity were measured as an initial screening output for evaluating cellular viability. Using the multiplexed capabilities of flow cytometry, we performed secondary and tertiary assays to further characterize the possible mechanism of action by analysis of cell cycle, specific RNA expression levels, oxidative stress, and caspase activation across differing concentrations of the compounds. This method and analysis highlights the complicated nature of assessing toxicity in cellular screening assays and the advantage of Flow Cytometry in particular for characterizing cell health.