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Flow Cytometry FAQ's

Cell Sorting

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How many cells do I need bring to the sorter?
This is the most commonly asked question. To answer it we need to know the following information:

  1. What is the approximate percentage of the population(s) you wish to sort?
  2. How many total cells do you want back?
  3. Do you want stringent purification or enrichment?
  4. How fragile or large are your cells?

All of these parameters influence the yield and purity of cell sorting. Here is an example: Let's say the subset you want sorted is 20% of the total and you need 2 million cells for your experiment. In theory you would need to run 10 million cells through the sorter (10 million x 20% = 2 million). However, the actual yield is usually 75-95% of this theoretical yield, due to abort rates (caused by sort conflicts) and also the quality of your sample. Therefore, we recommend that you bring 25-50% more cells to the sorter than you would need if the actual yield were 100% (based on the abundance of your target cells).

How long will it take?
The answer to this question depends primarily on your goal (enrichment vs stringent purification), the nature of your cells (fragile cells or large cells need to be sorted at lower pressures and speeds with a larger nozzle), and the concentration of your sample (more dilute samples will take longer to sort).

For example, on the MoFlo cell sorter, we use the 100µm nozzle at 30psi. For primary lymphocytes we run samples at maximum flow rates of 10,000-13,000/second. For cell lines, we run samples 400- 4,000/second depending on the size of the cells. With the Aria cell sorters, we can run primary lymphocytes at 6,000-8,000/second with the 100 µm nozzle at 30psi, or at 9,000-10,000/second with the 70 µm nozzle at 60psi.

Again, for cell lines the flow rates will vary a lot depending on the size of cells. For example, if your cells are run at 10,000/second, it would take approximately 30 minutes to run a total of 18 million cells through the sorter. Using the above scenario, your theoretical yield is 18 million x 20% = 3.6 million. If you require stringent purity or your sample is of poor quality (low viability, clumping, etc), your actual yield will be lower.

Do I need to filter my cells?
It is REQUIRED that your cells be filtered through at 70um nylon mesh, preferably right before running on the sorter. We can provide the mesh if necessary.

What do I re-suspend my cells in for sorting?
After the final wash, cells should be re-suspended at concentrations of 20-30 million (for primary cells) or 5-10 million (for cell lines) per mL in FACS Tubes (see below). The media can be any buffered isotonic salt solution (such as PBS) but we recommend Hanks Balanced Salt Solution (HBSS) calcium and magnesium free containing 10 mM HEPES, pH 7.2). Most cells benefit from having some protein in the media, such as 2% FCS, calf serum or BSA. However, serum concentrations >5% can lead to cell aggregation and clogging. For cell line samples, we recommend also adding 0.5% EDTA.

What are FACS Tubes and where do I get them?
BD Falcon makes FACS Tubes of various kinds. For all cell sorters, order 5 ml sterile polypropylene tubes (catalogue number BD2063). For non-sterile and analytical samples order catalogue number BD2002 (polypropylene) or BD2052 (polystyrene).

What kind of tubes or plates should the cells be sorted into?
For abundant populations we recommend 50mL (MoFlo) or 15mL (FACS Aria) Falcon Tubes, or 5mL Falcon tubes. We can also sort into a range of microtiter plates, including from 96-well to 8-well, microscope slides or Petri dishes. Please talk to us about your requirements and we will advise you on the best choice for your experiment.