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Lymphocyte Separation Media
High Performance Density Gradient Media for Mononuclear Leukocyte Separation
Isolation of viable mononuclear cells from blood serves as the starting point for a wide spectrum of immunology and cell biology studies. Early methods for isolating leukocytes were often unsatisfactory as the cells would become trapped by, and sedimented with, the aggregated erythrocytes, resulting in broad spectrum damage to the isolated cells. In 1968, a more convenient and rapid separation was introduced using centrifugation through a Ficoll-sodium metrizoate solution. This separation method takes advantage of cell density differences of the components in whole blood that, when centrifuged in the presence of a density gradient media, exhibits a unique migration pattern through the medium, allowing distinct cell populations to be fractionated. The whole process is performed at a lower gravitational force field without the introduction of aggregating reagents, thus preserving cell viability.
MP Bio offers two products for the isolation of mononuclear cells from human peripheral blood as well as bone marrow, and umbilical cord blood. Lymphocyte Separation Medium (LSM™) and LymphoSep® have been used for many applications by researchers worldwide.
1. Mononuclear Cell Isolation for Research Use Only
The optimized Lymphocyte Separation Medium (LSM) produced by MP Bio, has a unique formulation where sodium diatrizoate is successfully substituted for sodium metrizoate. LSM allows for the separation of lymphocytes not only from human peripheral blood, but also from bone marrow as well as umbilical cord blood. It has a density of 1.077-1.080 g/mL consisting of 6.2 g Ficoll and 9.4 g sodium diatrizoate per 100mL of solution. Figure 1 summarizes the separation principle when using LSM to isolate lymphocytes from the other blood constituents. This time tested and proven separation medium has over 2,200 scientific publications and was designed to ensure:
- Maximum yield of mononuclear cells
- Easy one-step centrifugation
- >96% cell viability of lymphocytes
- Low endotoxin
- Autoclave sterilization
2. Lymphocyte Separation for in vitro Diagnostics
LymphoSep lymphocyte separation medium is based on the highly optimized BØyum formulation, which was originally designed for the in vitro isolation of lymphocytes from diluted whole blood. It has a density of 1.077 g/mL. LymphoSep is validated for In Vitro Diagnostic (IVD) usage and has designation as an FDA class I exempt medical device for lymphocyte separation (21CFR864.8500). Similar to Lymphocyte Separation Medium (LSM), LymphoSep provides high yield of lymphocyte cells with more than 96% cell viability in one-step centrifugation.
Defibrinated or heparinized blood specimens are first diluted with physiological saline or balanced salt solution in 1:1 proportion, layered over the separation medium, and centrifuged at a low speed about 400 g for 15-30 minutes. During centrifugation, differential migration of blood constituents results in the formation of several cell layers.
The following layers will be visible in the conical tube, similar to that observed with LSM (Figure 1), from top to bottom: blood plasma and other constituents, a layer of mononuclear cells called buffy coat (PBMC/MNC), separation medium followed by a pellet at the very bottom which contains granulocytes erythrocytes (red blood cells). To isolate PBMCs, the buffy coat is extracted, washed with salt-buffered solution, and then centrifuged allowing the cells to be recovered with high yield in a small volume. The supernatant, containing platelets, separation medium, and plasma, is removed, leaving a pellet of purified PBMCs. These cells can then be used in life science applications, such as flow cytometry, cell sorting, cell culture, and sequence etc.