This article aims to explain the 4 different parameters you can adjust when using the LeviCell for live cell enrichment.
LeviCell technology together with its simple workflow, offers a good viable cell enrichment for most cells using the standard recommendations for levitation agent concentration (150 mM, final) and 20 min of levitation time. This approach requires no cell staining, labeling, or use of cell surface antibody markers. The gentle process virtually eliminates undue stress on cells and allows users to ultimately analyze their cells as close to their in vivo state as possible.
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There are four factors that can be adjusted for a successful LeviCell run: cell number or concentration, levitation agent concentration, levitation time, and split line. These factors can be tweaked to achieve optimal results for cell enrichment without the need for staining or labeling. Changes to the above factors can be summarized as follows:
Levitation Agent Concentration
One of the main parameters that users can, and often should, optimize is the concentration of Levitation Agent (LA) in the final Levitation Buffer containing the cells. Levitation Agent is supplied at a stock concentration of 1M and should be added directly to the Levitation Buffer containing cells at a final concentration between 50 mM to 150 mM (<50 and >150 in rare cases). In general, the concentration of LA is directly related to the levitation height of the cells within the magnetic field:
By decreasing Levitation Agent concentration, cells will levitate in a lower position. In addition the bands can broaden, potentially decreasing the resolution between the live cells and the dead cells.
Cell Concentration
The total number of cells loaded into the cartridge, for a given cell type, can determine the levitation behavior and ultimately how the bands appear at final levitation height. Generally, when testing a new cell type, it is best to start with a total cell input of 250,000 cells and avoid using more than 1 million cells. With this number of cells, a live cell and dead cell band can be distinguished. Clear bands can be seen as well when higher cell numbers are loaded, and the bands do appear broader as a greater number of live and dead cells are added.
Additionally, cell size and cell viability should be considered as they can influence levitation behavior.
Cell size – given that the levitation chamber has a finite volume, larger cells will occupy more space than smaller cells, therefore the user should consider using fewer cells when cell size is > 20 μM.
Viability – determining the levitation behavior of viable cells requires that enough viable cells are introduced into the cartridge to visually confirm where they levitate relative to the accompanying dead cells and debris in the cell suspension. If viability is very low (e.g., < 30%), it may require more than 250,000 total cells input to visualize the live cell band adequately and to have a good recovery of your viable cells.
Levitation Time
The amount of time required for cells to reach their final levitation height varies. Larger cells will reach equilibrium very quickly, often within 5-10 min, especially if cell size exceeds 20 μm. Most cells fall within the range of 5-20 μm and require up to 20 min of levitation time. Cells smaller than 5 μm often need 30-40 min of levitation time. The LeviCell Experiment Manager software comes with 3 different protocols based on the size of the cells. The pre-programmed times are: 40 min for cells <5 μm; 20 min for “standard” cells 5-20 μm, and 5 min for cells > 20 μm.
Split Line
Where the split line is set can define the quality of the output samples. Setting the split stringently can maximize the purity of the lives cells ensuring highest viability however the tradeoff is a potential drop in yield. Setting the further away can maximize yield and may or may not affect the purity of the viable cells.
If a high purity of viable cells is most desired, the split line may be set very close to the bottom of the viable cell band. Setting the split line very close to the viable cell band will ensure capture of predominantly viable cells, and because the cells are homogenous, there is no risk of inadvertently excluding a specific cell type from capture. If yield is most important for the downstream workflow, the split line may be set further below the bottom of the viable cell band to ensure collection of the maximum number of viable cells.
It is important to recognize that selecting for purity or yield can be a trade-off. Choosing purity over yield will result in some fraction of viable cells separated into the waste outlet of the cartridge. On the other hand, choosing yield over purity will result in maximum capture of viable cells in the sample but may also capture a few cells that, while “live/not dead”, are not fully viable (e.g. apoptotic) and are levitating just below the live cell band.