Processing and staining cells for flow cytometry or cell sorting is a multi-stepped process that can stress cells and cause cell death. Dead or apoptotic cells are troublemakers in cytometry because they can bind antibodies, effectively changing the staining antibody concentration even after titration, which results in suboptimal staining of live cells, and they can autofluoresce, particularly in the green channels, providing false positive signals. Dead cells also do not divide, cannot produce cytokines upon in vitro stimulation, and are often sticky due to extruded nucleic acids resulting is both poor yield and poor purity when sorted. So if you have a large fraction of dead cells in your samples, your results may be significantly skewed or unusable.
Flow Cytometry Blog
Flow cytometry is a valuable research tool because it can generate abundant amounts of very detailed data, but this often means that scientists need to deal with moving and storing large amounts of digital data. In addition, many flow cytometry users run their experiments at a site separate from their lab or work with an offsite contract research organization (CRO) that runs their flow cytometry experiments. This means that flow cytometry data transfer is an issue that researchers must address even in the planning stages of their flow cytometry experiments. Consider these factors related to transferring large datasets for flow cytometry.
Posted in Data Analysis
We walked you through the basics of achieving your optimal panel design in the “Know your Flow” series, but you probably have more questions as you refine your panel or stumble upon some of the technical nuances of staining cells.
Check out these tips for a successful flow cytometry run.
Posted in Preclinical Development
Flow cytometry analysis only provides usable data if you analyze living cells. In both preclinical and clinical studies, including a live/dead stain in flow cytometry panels is essential for discriminating live from dead cells in order to analyze only live cells. Live/dead stains are also called viability dyes, and different types of dyes use different chemistry to stain live or dead cells. Consider these factors when including a live/dead stain in flow cytometry panels.
The word “validation” strikes fear and anxiety in the hearts of many researchers crossing into the world of flow cytometry for clinical trials. Validation plans evaluate assay reliability and overall performance and may include but are not limited to measuring precision, robustness, sample stability, assay specificity, and intersample variability.
Many research scientists working in basic research settings haven’t had to consider how to translate a basic flow cytometry protocol into a validated protocol and may think this is such an overwhelming process that they should avoid clinical trial research altogether. However, any well-trained scientist can develop a validated flow cytometry assay, especially with the assistance or collaboration with scientists who have validation expertise.