High-quality Flow Cytometry analysis is dependent on the preparation of high-viability, single-cell suspensions. In this regard, peripheral blood and suspension cell cultures require minimal processing; however, solid tissues such as the spleen, lymph nodes, skin, neural samples, and solid tumors require efficient dissociation and processing to form single-cell suspensions before analysis. Robust and effective standardized methods for tissue dissociation involve either mechanical dissociation, enzymatic digestion, or a combination of both to ensure high yields of single-cell suspensions with high viability for flow cytometry applications.
Expertise in mechanical/enzymatic and combination approaches to tissue processing and dissociation- ensures optimal recovery of high- viability, single-cell suspension.
Standardized Methods for the processing of a range of complex tissue types including skin, bone marrow, spleen, lymph nodes, neural tissue and tumors.
Quality Control to ensure high viability and cell recovery, mitigation of sample clumping, and preservation of protein epitopes.
Optimizing the dissociation process for specific tissue and target epitopes can be a trial and error process. FlowMetric has a library of robust protocols for the optimal processing of a wide variety of tissue types, ensuring maximum viable cell recovery while minimizing cell clumping and epitope loss through the side activities of enzymatic processes.
FlowMetric offers customized Mechanical Dissociation protocols utilizing a blender-type mechanism with the GentleMax™ system. Optimal tissue processing starts with the selection of transport buffer or media, and transport temperature, along with the removal of as much connective tissue and adipose as possible. Mechanical dissociation is a rapid technique that works well on samples that are loosely associated with tissues such as mouse spleens, bone marrow, and lymph nodes.
Alternatively, FlowMetric offers several optimized enzymatic dissociation protocols that employ a combination of proteolytic processes using enzymes such as collagenase and/or trypsin, as well as Deoxyribonucleases (DNase) to reduce cell clumping. There are many commercially available proteases, with varying levels of potency. The goal of enzymatic dissociation is to disrupt the tissue structure effectively enough to maximize cell yields and viability, but without damaging the epitopes on the cells. This can take some optimization and may involve using a combination of different proteases. Weakly digestive proteases include Dispase™ and Collagenase type 3, whereas Trypsin, Papain, and Elastase are strongly digestive and dissociations using these enzymes should be carefully monitored. Liberase and Blendzymes are commercial protease cocktails that are optimized for different tissue types. It is important to understand the structure of complex tissues in order to effectively dissociate the connective tissue and maximize the yield of high-viability cells. For example, the effective dissociation of cells from skin tissue involves an initial treatment with Dispase™ to separate the epidermis and dermis layers, and then collagenase to breakdown the extracellular matrix and enable trypsin to disperse the tightly associated cells including the keratinocytes and keratinocytes stem cells, the dermal derived fibroblasts and the mesenchymal stem cell-like cells This process is summarized below.
Once optimized, enzymatic digestion can be very effective, efficient, and reproducible. However, it is important to note that enzyme dissociation can modify proteins on the cell surface, which can alter cell function or binding of fluorescent antibodies, so the FlowMetric team recommends that each process should be validated for every flow cytometry panel.
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. Our experience in specimen handling and practical know-how will help to ensure the recovery of even the rarest cell populations from complex tissue structures.
From immunophenotyping Tumor-Infiltrating Lymphocytes recovered from solid tumors, to skillfully preparing highly viable, single-cell suspensions for sorting applications, our team processes every sample, for every study with a commitment to quality. Please contact us to discuss your program needs with our scientific team and learn more about how FlowMetric can support and accelerate your research and clinical trial initiatives.