Instruments - Sorting

Fee Structure:

Sort rates are: $30 setup fee and $90 an hour billing, with a 1 hour minimum. Time is then billed to the ½ hour. Time billed will be time reserved, unless you run long, then the extra time will be added to the bill. Bills are sent out at the end of the month.

Please direct all billing questions to Tim directly.

Aria Guidelines

A sort form is required within 24 hours of registering for time -- sort time will be canceled if this form is not completed.  It is vitally important that the entire form is read and questions answered.  Biosafety information will be verified with the IBC.

Sorts canceled less than 48 hours before sort time will be charged for the time reserved.

New investigators are required to schedule an appointment to discuss their sorting needs with me.

Key things to remember:

• When scheduling time, please plan on an extra 1/2 hour for instrument setup. If scheduling a sterile sort, schedule an extra 1/2 hour (1 hour total).
• Before a sort will be started, the sort logic will be reviewed. Please make sure you are available for this review. Failure to review the sort logic will result in the sort being canceled and the user billed for the scheduled time of the sort.
• After the sort is started, provide the operator with contact information so that you can be contacted if something arises during the sort, or for notification of completion of the sort.
• Samples must be filtered.
• Samples can be accepted in 1 ml, 3 ml, and 15 ml tubes.
• Samples should be at 50x10e6 cells/ml for high speed sorts, and 25x10e6/ml for low and medium speed sorts.

Aria Configuration: 

488 Laser  || 633 Laser  || 407 Laser

488 LASER:

633 LASER

407 LASER:

SORT SPEED AND CELL SIZE

The Aria has two nozzle sizes at present, 70 micron and 100 micron. For good stream stability, cells being sorted should be no more than 1/4 to 1/5 the size of the nozzle. This gives the Aria an effective limit on sorting cells below about 50 microns in diameter.

Furthermore, the stability of the stream is related to the nozzle size. Using a 70 micron nozzle, the system is stable at 70 PSI (the 'high pressure sort option'), and for most cell types this is the best place for sorting. If you have a small, fragile cell, the pressure can be reduced to 35 PSI (the 'medium pressure sort option'). For larger cells such as adherent cell lines, the use of the 100 micron nozzle at 20 PSI (the 'low pressure sort option') is best.

Together, this information can be used to determine the ideal cell concentration to get a reasonable sort rate.

When you bring your cells to the sorter, have your sample at close to this sample concentration. If it is more concentrated, it can be diluted, so bring some of your buffer as well.

ADHERENT CELLS

Adherent cells pose a special problem for a sorter. In general these cells have been treated with trypsin to remove them from the culture plate. Standard practice is to then add serum to neutralize the trypsin. However, this causes problems, as trypsin adds back diavalent cations necessary for cells to adher to each other. It is better to use a trypsin inhibitor to inactivate your trypsin rather than serum. Adherent cells, activated cells and cells with extensive structures outside the cell membrane all benefit from a slower sort.

DNAse in SORTING

As cells die, they can release their DNA into the sample buffer. This released DNA readily binds to other cells, causing clumping and reducing the effectivenss of the sort. The addtion of 10 Units of DNAse per ml of sample will help prevent this effect. This is especially important with adherent cells.