Ideal Cell Suspension Concentrations

Having the sample too concentrated, or too diluted can be problematic.  There is no ideal concentration that works for all cell types and sort set-ups.  It is a matter of understanding some of the issues and deciding what factors are most relevant to a given cell type and experimental design.

  1. Coincidence Aborts:  While the sorter is evaluating which cell to sort, it must also determine whether it can do so it a manner that ensures the sorted material remains pure.  The sorter makes this decision based on the proximity of events in time.  If the desired event is too close to a potential undesired event, the machine will abort (not sort) the desired event to ensure purity.  There are other modes of sorting that favor recovery over purity, so if total cell numbers are more important than purity, we can accomodate this.  This should explain why we would not want the sample so concentrated that it becomes difficult to space the cells far enough apart while they are going through the sorter.  If recovery is a prime concern, aborts tend to be the issue.
  2. Signal CVs and Sensitivity:  If the sample are too dilute, to get them to run at a reasonable rate can cause another set of problems.  To get them to run at the appropriate rate, the sample differential must be increased.  If the differential is too high, the CV's (coefficients of variation) start to become higher.  This ultimately leads to less resolution and lower sensitivity.  If you are trying to separate two very close populations, the CV becomes more important.
  3. Cell Adhesion and Clumping:  Adherent cells are trickier to sort than suspension cell types.  Adherent cells typically like to stick to each other given the ooportunity.  Careful cell preparation and media can help avert this problem, but so can cell concentration.  These sticky cell types typically like to be slightly more dilute to mediate clump formation.  There is no absolute formula for this, it is determined by trial and error for a given cell type.  The take home message is to consider running very sticky samples slighty more dilute than the below suggestions.

The following is a list of concentration ranges based on machine set-up which typically correlates with cell type:

Nozzle Size

Cell Types

Concentration (per mL)

70um

lymphocytes, thymocytes

8-12 x 106

80um

activated subsets, smaller cell lines

7-9 x 106

100um

larger adherent cells

5-8 x 106

 

These guidelines are in place to suggest where to start with, but it might be easier to tend towards a little too concentrated (towards the high end of the recommended range), but bring some additional sample buffer with you so things can be diluted as necessary.

 

questions or comments: asaluk@scripps.edu