Flow cytometry analysis is a critical technique for guiding preclinical drug development. Flow cytometry data can provide mechanistic insights and define potential biomarkers that correspond to clinical outcomes. But getting the most out of your preclinical flow cytometry analysis may hinge on how your cell samples are handled, specifically whether you are working with fresh cells or fixed cells.
Flow Cytometry Blog
What is flow cytometry? Flow cytometry is a semi-quantitative technique that allows you to analyze the frequency and other properties of cells stained with specific fluorochrome-conjugated antibodies. Flow cytometry is most frequently used to monitor the immune response due to the fact that the frequency and functionality of different immune cell subsets can be measured concurrently. Flow cytometers are the instrument at the center of this technique and have evolved over the past few decades to become an essential instrument for biomedical research.
Flow cytometry assays can only deliver reliable, high quality data if the cells used in the assays are viable and handled properly. Immune cells, including lymphocytes and dendritic cells, can be isolated from whole blood using density gradient centrifugation protocols. These peripheral blood mononuclear cells (PBMCs) can be used as fresh cells for flow cytometry or can be frozen for later use. Consider the pros and cons of using each type of cell preparation when you are planning your next flow cytometry assay.
Intracellular staining (ICS) assays are one of the most commonly used flow cytometry assays in basic and clinical research. These customizable assays allow users to measure various cytokine responses in different immune cell types. But why go through all this effort when ELISA or bead-based assays can also measure cytokines? Check out these three features of ICS assays that make them stand out from similar cytokine assays.
High quality flow cytometry assay data can be obtained with well-maintained flow cytometers and high-quality processing and staining protocols. At the center of these operations, are the inclusion of the appropriate control samples, which is critical to guaranteeing the quality of flow cytometry assay data. Isotype controls and fluorescence minus one (FMO) controls are critical in assuring that the correct fluorescence signals are being measured, and also that these controls are separate from compensation controls which address spectral overlap effects. Consider these aspects of FMOs and isotype controls as you plan your next flow cytometry experiment.