How many cells should You bring?

The quantity of cells required is influenced by two key factors:

1. The number of cells needed for subsequent tasks.
2. The rarity of your cell population within the pre-sorted sample. By gaining a rough understanding of these variables, you can estimate the necessary cell count.

It's advisable to bring approximately 20% more cells than your initial estimate to account for potential inaccuracies. Factors contributing to this margin include:

• A percentage of the pre-sorted sample may be non-viable (unsuitable for sorting).
• Cell counting methods inherently possess a level of inaccuracy.
• The actual cell count in the pre-sorted sample might be lower than anticipated.

If your downstream application requires a minimum cell count, allow for a 15-20% margin of error within the sorted population, as the sorter's cell count is, at best, 85% accurate.

When bringing your cells, use FACS tubes with a buffer that best suits the cells, or alternatively, use an Eppendorf for ImageStream analysis. Ensure the protein concentration in the buffer does not exceed 2%.

For conventional Flow Cytometry or Sorting, maintain a cell concentration of no more than 2x10^7 cells per ml, while ImageStream analysis requires a minimum of 1x10^7 cells per ml. Bring extra buffer to the sort in case dilution is needed.

To prevent instrument clogging, filter your sample. A general rule is to use a filter size about half that of the sort nozzle. For instance, cells sorted through a 70 um nozzle should be filtered through a 30 um filter.

Specify the collection details before the sort setup to prevent delays.

It is recommended to collect cells into a buffer to avoid stressing the sorted sample. The larger the volume of the collection buffer, the less stress on the cells, though it may result in collecting fewer cells per tube. Consider collecting cells into neat FBS/FCS or high-concentration FBS/FCS cellular media to enhance the survival of the sorted sample.