Cell Sorting By FACS
SP, Nov 2005
Currently, the Moflo instrument (Dakocytomation) is used to sort cells at the AECOM FACS facility.
Sorting is performed by the person in charge at the facility.
You need to book an appointment in advance. (at calendar.yahoo.com, use aecom_moflow / facs**** to log in).
Stain cells for FACS as usual but under sterile conditions (unless you don't need to grow the sorted cells). Note that certain staining procedures may kill cells (e.g., using glucose-free buffers, staining with certain lectins, incubating too long, etc.).
Finally, resuspend the cells in HBSS or serum-free medium with pencillin, streptomycin and Fungizone™ (or a similar anti-fungal agent).
When you go to the facility, take your sample on ice, and also take twice as many (as samples) collection tubes (sterile 12 x 75 mm polypropylene tubes like Falcon 2063) containing 2-3 ml of HBSS or serum-free medium with pencillin, streptomycin and Fungizone™ (or a similar anti-fungal agent).
Requirements of the FACS facility at Einstein
0. Samples should be more than 0.5 ml in volume with density of less than 15 x 10e6/ml but otherwise as high as possible.
Use 12 x 75 mm polypropylene tubes. Other tubes will not fit or their walls may crack because of pressure.
1. Cells MUST be in a single-cell suspension.
If you removed adherent cells by trypsinization, etc., resuspend them well (observe under microscope). Cell clumps should also be removed by passing cells through a cell-strainer. Note that there are reagents such as Accutase™ that are better and less cytotoxic than trypsin. Also, 5 mM EDTA in the sample will prevent cells from clumping. Many times, large number of dead cells can cause clumping through the released genomic DNA; this can be prevented by adding DNAse to 10 U/ml.
2. Cells can be in culture medium with serum. However, medium is not an ideal buffer for sorting because pH indicators such as phenol red can cause emission interference and calcium in the medium can be precipitated by phosphates in buffers used in the sorting machine.
A basic sorting buffer is cation-free PBS with 1 mM EDTA containing 25 mM HEPES at pH 7 and 1% serum.
3. The default nozzle used by the machine has a 100 um opening. If your cells are more than 20-25 um in size, please notify in advance.
4. Note that cells can be sorted into 12 x 75 mm tubes, into 15 ml or 50 ml conical tubes, glass slides, 24-well plates, 96-well plates, etc.
5. You may need to bring in a control sample (like in regular FACS) for adjusting the machine settings.
6. Please inform beforehand if you are using biohazardous substances.
7. Do not underestimate sorting time.
It will take a minimum of about 5 minutes to sort to get 100,000 cells representing 1% of a sample provided at highest density possible (15 x10e6 cells/ml). Add time to adjust the machine, set up collection tubes, etc.