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).

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.

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.

Proper sample preparation is absolutely essential for obtaining good purity and yields. 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 1-2% FBS, calf serum or BSA. Higher serum concentrations can lead to cell aggregation and clogging. For cell line samples, we recommend also adding 1mM EDTA. We recommend using buffers that do not contain phenol red because phenol red causes an increase in background fluorescence, and reduces sensitivity. This effect is particularly bad for dim green signals.

For sticky cells:  Increase the concentration of the EDTA to 5mM and use FBS that has been dialyzed against Ca/Mg++ free PBS or 0.5% BSA. Some cell types, however, can be sensitive to high concentrations of EDTA.

For adherent cells:  To detach adherent cells, trypsin (or other detachment buffer) is often quenched with media or buffer containing serum.  This reintroduces cations that can promote aggregation. Use FBS that has been dialyzed against Ca/Mg++ free PBS or increase the EDTA concentration to 5mM or higher (Some cell types, however, can be sensitive to high concentrations of EDTA.). Accutase and Accumax are cell detachment solutions that can be used to generate single cell suspensions.

NOTE: Accutase can alter some cell surface epitopes; the effect will need to be determined empirically for the epitopes being evaluated.

For samples with a high percentage of dead cells:  DNA from dead cells can cause cell clumping. DNAse I in the presence of magnesium chloride will help reduce cellular aggregation.

1. Treat cells for 15 to 30 minutes in a solution of 100 µg/mL DNAse and 5 mM MgCl2 in HBSS at room temperature.
2. Wash the cells once in HBSS with 5 mM MgCl₂.
3. Gently resuspend the cells in HBSS with 5 mM MgCl₂ and add 25-50 µg/mL DNAse (as a maintenance dose) prior to and during the sort.

Note: DNAse I requires at least 1 mM magnesium to work effectively, although 5mM is optimal. It is important to minimize the presence of dead cells during this procedure, since actin released from dead cells irreversibly inhibits DNAse I.

You will need to bring an unstained control, single colour compensation controls and you MAY need to bring FMO (Fluorescence Minus One) gating controls. You may want to consider an FMO control if you are using more than 5 colours simultaneously and if your populations of interest cannot be discretely separated from background/negatives.

Please refer to the table below to see an example of a typical sort experiment with three colours.

Fluorophores excited by the Yellow/Green laser
If you are using markers such as mCherry, RFP, Tomato Red, Texas-Red, etc, Please contact the Flow Facility to inquire about the specific sorter to book as only a limited number of sorters are equipped with an appropriate laser to detect these signals.

Viability Dyes
We recommend that you add a viability dye such as PI, 7AAD, or LIVE/DEAD fixable dyes (Invitrogen) to your sample to enable exclusion of dead cells during sorting. If you choose to do so, please bring an appropriate single color compensation control. We suggest the following: Heat killed cells (65oC for 15 minutes and then mix with live cells) followed by staining with viability dye.

For abundant populations we recommend 50 mL (MoFlo) or 15 mL (FACS Aria) Falcon Tubes, or 5 mL 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.

We recommend 100% serum or HEPES buffered HBSS.

The sorter uses PBS as its sheath fluid. Mixing large quantities of PBS with buffer containing calcium chloride may produce a precipitate if calcium phosphate on the cell membranes. This can adversely affect viability. For this reason, it is preferable to sort large numbers of cells into 100% serum, and not into regular culture medium. HEPES buffered HBSS without calcium is an alternative. If you must sort into medium, remember that the pH will rise with time, and it’s best to start with it on the acid side of neutral.