In a healthy person, immune cells like B cells, T cells, and macrophages are typically surveilling the body for abnormal cells or infectious agents. These cells don’t fire up their inflammatory toolbox unless they recognize one of these foreign entities. The potent inflammatory mediators activated during these responses include cytokines, free radicals, prostaglandins, and clotting factors, which must be tightly regulated to avoid wreaking havoc on healthy tissue. This exquisitely controlled activation of inflammatory molecules means that when you look for them in cells by flow cytometry, they may be very difficult to detect.

The immune system is comprised of a multitude of unique cell subsets. Each cell type, from B and T cells, to macrophages, monocytes and dendritic cells, have been phenotypically subdivided into unique subsets as we learn more about the phenotypic signatures that define these cells. Flow cytometry has been the central tool in evaluating and defining cell subsets, and major advances in immunophenotyping have occurred recently as more parameters can be measured during a single run on newer flow cytometers.

Immunotherapeutic molecules currently being used in the clinic are powerful immune modulators, but their effectiveness can be inconsistent between patients. Clinicians and scientists use different assays to evaluate why immunotherapies fail in the clinic. The flow cytometry-based receptor occupancy (RO) assay is a critical tool for evaluating the effectiveness of immunotherapies in the clinic. Here are three features of flow cytometry-based RO assays that give them clinical value.

The idiom, garbage in, garbage out applies to many areas of scientific research, including flow cytometry. Good sample preparation is critical to accurate and sensitive cytometry analysis of cells, wherever their origin. 

Q is for Quality - QA, QC and Flow Cytometry How do clinical flow cytometry labs ensure that the data they generate is accurate, reproducible, and conforms to regulatory requirements? They use quality management systems, including quality assurance (QA) and quality control (QC). Some scientists seem to use these terms interchangeably, but what do they really mean and why are they important to flow cytometry?

For most applications flow cytometry is used to identify cell populations and define bivariant terms of positive and negative sub-populations according to specific biomarkers, through the binding of fluorescently tagged monoclonal antibodies (mAbs). Typically, the cutoff between these populations is set relative to a control unstained population. Since the fluorescent intensity of a signal is proportional to the amount of monoclonal antibody bound to that cell target, this signal is directly related to the expression level of that target. However, for flow cytometry endpoints to be considered truly quantitative and fulfill the rigor of clinical utility, several obstacles needed to be overcome. In this blog, we explore the rationale behind quantitative flow cytometry, and the tools that are now being implemented to help achieve standardization.

What are receptor occupancy assays?

Introduction of CAR-T Therapy T lymphocytes are engineered with synthetic receptors known as chimeric antigen receptors (CAR) in CAR-T Cell therapy. The CAR-T cell is an effector T cell that recognizes and eliminates specific cancer cells, independent of major histocompatibility complex molecules. (Zhai et al. 2018). Chimeric antigen receptors (CARs) cells have recombinant receptor constructs expressed in T cells to target cells expressing specific antigens.

Flow cytometry is a powerful tool for surveying the cellular landscape during preclinical development of drugs and biologics. But flow cytometry can go beyond immunophenotyping to actual functional measurements that can contribute to understanding the true potential of a therapeutic candidate. To make the most of your flow cytometry studies, consider these other assays as you plan the next phase of preclinical development.

Flow cytometry is a powerful tool because it allows users to analyze the characteristics of millions of cells with relative speed and precision. A single cell suspension of fluorescently labeled sample travels through the cytometer for excitation by lasers, and the emitted light photons are measured by different detectors. Having a single cell suspension is essential to measuring cell fluorescence accurately, and many types of cell or tissue samples must be specially processed to make this suspension. Two different methods can be used for single cell suspensions: mechanical dissociation of tissue or enzymatic dissociation of tissue. This processing step is typically carried out before cells are stained and both methods have benefits and caveats.

Memory is a characteristic of the immune system that provides humans and other vertebrates with long term protection against infectious diseases and other “non-self” antigens such as those associated with tumor cells. In the context of T cells, memory responses occur when a naïve T cell encounters an antigen bound to a major histocompatibility complex molecule and is activated to undergo differentiation into an effector cell or a memory cell. Memory T cell populations can persist in the body for months to years and can be stimulated to respond specifically and rapidly to a foreign antigen upon re-exposure.

Flow Cytometry utilizes fluorescently labeled antibodies to detect specific biomarkers on the surface and within cells, and over the past few years, there has been a surge in reagents available for flow cytometry applications. Most of these have been developed using monoclonal antibodies raised in mice and conjugated to a range of fluorophores. However, there are still instances where suitable monoclonal antibody reagents/conjugates are not commercially available, and small-scale conjugations are not practical. In these instances, so-called indirect staining may be employed, where the binding of an unconjugated primary antibody is detected using a secondary anti-IgG antibody conjugate.