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Preparing a single cell suspension for Levitation

The LeviCell enrichment process involves isolating live cells from a single cell suspension. To optimize levitation results, several factors need to be considered.

  • What is the total cell concentration, and viability, of the single cell suspension?
  • What is the debris burden in the suspension?
  • Are there clumps of cells that are not fully dissociating into single cells?
  • Are you working with cells from tissue / tumor or cells that naturally exist in suspension?
  • Would depleting certain cell types be beneficial - i.e utilizing the LeviSelect kit?
  • Is your cell suspension contaminated with Red Blood Cells (RBC's), and is an RBC removal step needed?

Tissue dissociation, cellularity and debris

Since each biological tissue type is unique, there is no one-size-fits-all tool for achieving a good single cell suspension. It is essential to determine the most effective tissue dissociation protocol based on past experience. The tissue dissociation protocol will determine the final cellularity, viability and debris content of the suspension. Further, during levitation, cell clumps remain in the sample and levitate at their average equilibrium position. Clumps can reach their levitation equilibrium within the live cell band. Therefore, one or more filtration steps may be necessary.

Pro Tip: For most tissue dissociates, a single filtration step through a 70 μm filter can enhance levitation output. In cases of particularly contaminated samples containing high quantities of ECM or calcified material, a second filtration step through a 40 μm filter may be beneficial.

When loading a single cartridge in the LeviCell 1.0 or a lane in the LeviCell EOS cartridge, the optimal number of cells to load depends on the amount of debris present. In samples with high debris levels, higher viability and purity of the output sample can be achieved with a lower initial cell load . Excessive debris during levitation can negatively impact the process. As a general guideline, samples with high debris should be loaded with no more than 500,000 total cells, while cleaner samples can accommodate multiple millions of cells in a single run.

Cell selection and clean up

For your tissue of interest, it may be that you are not interested in all the cell types that you would obtain upon dissociation. In this case, you may be looking into strategies to remove certain cell types - e.g removal of red blood cells, or removal of the immune (CD45+ ) fraction. It may be that in your current workflow you utilise technologies such as magnetic beads, columns, or a hypotonic solution. When preparing a suspension for the LeviCell, you may want to think whether these steps are necessary, or whether alternatives can be used such as depleting these cells types with our LeviSelect kits. In this case, it maybe that you can save time by skipping a processing step, and instead replacing it with a simultaneous depletion during Live Cell Enrichment.