Guidelines for Getting a Good Sort

As of the May 1, 2012 all users that are requesting cell sorting are required to download the Cell Sorting Guidelines from the EHSO. A Risk Assessment must be provided to the Flow Cytometry Facility before any sorting can be performed, a template is provided within the EHSO guidelines.

Sort Calculator (Designed by Larry Arnold)

ECFCC - Aria II Filter Set

Cells – How many do I need?

To answer it we need to know the following information:

• What is the approximate percentage of the population(s) you wish to sort?
• How many total cells do you want back?
• Do you want stringent purification or enrichment with great recovery?
• Do you have large or fragile cells?

All of these parameters influence the yield and purity of cell sorting.

For 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).

Time – 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).

Filtering – Do I need to filter my cells?

It is REQUIRED that your cells be filtered through at 40um – 70um nylon mesh, preferably right before running on the sorter. We recommend the following tubes: Falcon® 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap, cat # 352235

Re-suspension – 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 10-20 million (for cell lines) per mL in BD 5mL 352063 Polypropylene tubes or 15mL BD 352196 Polypropylene tubes.

We recommend the following sort buffer for re-suspending your cells in:

1x PBS
2% FBS or BSA
25nM HEPES at a pH 7.0 (Stablizes Cell Membranes)
EDTA if your cells are sticky (1mM is usually fine)
10units/mL of DNASE if there is high cell death.

We highly recommend using a Live/Dead Stain when cell sorting.

Collection Media – What media should you sort into?

We recommend that you sort into culture media with at least 20% FBS. For RNA/DNA PBS and if you have cells that are very fragile FBS only.  Please discuss with the Technical Director for the best medium to sort into.