Tissue Dissociation & Sample Preparation
Flow Cytometry analysis requires single-cell suspensions, such as those found in peripheral blood or cells grown in suspension. Adherent cell lines, solid tissue samples, and tumors require tissue dissociation and processing into single-cell suspensions before they can be analyzed. Numerous protocols for sample preparation and tissue dissociation are available and may involve enzymatic digestion or mechanical dissociation of the tissue, or a combination of both. Mechanical disaggregation is suitable for loosely bound structures such as adherent cells from culture, bone marrow, and lymphoid tissue. Various techniques are employed, from a simple pestle and mortar to the repeated passing of tissues through fine gauge needles, and there are number of commercial systems such as the gentleMACS from Miltenyi that provide optimal tissue disruption.
For instances where mechanical disaggregation is not desired, proteolytic enzymes are used to disrupt protein-protein interactions and the extracellular matrix that holds cells together within a tissue. Protease activity is influenced by factors including pH, temperature, and the presence of cofactors. It is important to note that care must be taken when choosing an enzyme, as they may result in the destruction of the antibody epitope. For example, pepsin works optimally between pH 1.5 and 2.5, but the acidic conditions would damage cells if left for too long, and cell surface antigens of interest may be diminished or completely lost. There is a wide range of commercially available proteases for tissue digestion, from very strong enzymes such as papain, hyaluronidase and Trypsin, down to weakly digestive enzymes such as Liberase™, Dispase™, and collagenase type 3. Chelators like EDTA, can remove divalent cations responsible for maintaining cell function and integrity, but their presence may inhibit certain enzymes. For example, collagenase requires Ca2+ for activity. DNAse I is often employed to minimize cell clumping and can be added either at the beginning or end of protease digestion. The dissociation of neural tissue for flow cytometry analysis has additional complications. Typically, enzymatic digestion is performed using collagenase and phospholipase and the digested sample passed through a filter. It is then necessary to treat the sample with anti-myelin beads to capture the high levels of myelin protein present that can significantly block epitopes on the neural cell surface.
Selecting the optimal enzyme or enzyme combination is critical to ensure maximum cell yields and viability as well as the minimizing of epitope loss through the side activities of enzyme activity. In all situations, removing cell clumps, dead cells, and debris is essential to eliminate false positives and obtain results of the highest quality.
When preparing samples for cell sorting, it is critical that the cells maintain a high viability. Frequently, soybean trypsin inhibitor is employed to more effectively neutralize trypsin after digestion is completed. The cell suspension should be kept on ice in calcium and magnesium -free PBS to minimize cell clumping, and if aggregation is an issue then the addition of EDTA, DNAse I and MgCl2 can help resolve this. Filtering of samples through a Nitex Nylon filter prior to sorting is advised and the use of low protein sort buffer can help minimize autofluorescence. As with most flow cytometry analysis, the use of a live/dead stain is highly recommended when sorting cells from dissociated tissue samples.
Optimizing the dissociation process for a given tissue and target epitope is often a trial and error process. However careful handling and the adoption of some precautionary steps can improve viable cell yields from even the most complex tissue structures .
