Cells that have died or been damaged during the cell separation process spill their contents and expose the neighboring cells to an array of factors that could produce undesirable effects. Those effects could include changes in growth, activation of stress response pathways, initiation of cell death programs, and other alterations in their normal physiology that make obtaining meaningful data from downstream applications challenging. Therefore, the health of isolated cells is essential for ensuring success and is particularly important in cases where live cells are required. For instance, when assessing cellular function or simply culturing the isolated cells.
The most common way of assessing cellular health is by determining viability. Viability is merely a measurement of the proportion of isolated cells that are living after the cell separation procedure. Dyes that enable evaluation of membrane integrity are frequently used for this purpose. Membrane impenetrate dyes such as trypan blue, DRAQ7TM, 7-AAD, PI (Propidium iodide), or DAPI (4′,6-diamino-2-phenylindole, dihydrochloride) can only stain dead or dying cells with damaged membranes. Isolated cells exposed to these dyes can be evaluated with a flow cytometer, microscope, or cell counter to determine overall viability.
Viability is calculated as follows:
Using a quick and gentle cell separation method is a great way to ensure maximum viability. While magnetic activated cell sorting (MACS) and buoyancy-activated cell sorting (BACS) are among the quickest cell separation methods available, BACS represents a much gentler approach to cell isolation. Since MACS relies on much smaller particles than BACS, there is the potential that these particles could be taken up by target cells. Additionally, MACS relies on a strong magnetic field to pull the target cells away from the rest of the sample. The potential uptake of the magnetic particles coupled with the harsh forces associated with the strong magnetic field may lead to physiological changes that hide or distort the phenotype you are investigating.
CD19+ B cells removed from mouse spleens were analyzed before (left) and after microbubble isolation (right). Cultured isolates were exposed to CpG and IL5 for three days and then plasma cell induction (CD138 upregulation) was assessed (bottom). Data courtesy of Derek Jones (University of Pennsylvania).