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.
• For investigators who need to use the 130 micron nozzle, please reserve an extra 1/2 hour to allow for nozzle changeover. You will also need to reserve the 130 micron nozzle on the calendar.

Aria Configuration: 

488 Laser  || 633 Laser  || 407 Laser

488 LASER:

633 LASER

407 LASER:

SORT SPEED AND CELL SIZE

TThe default configuration of the FACSAria uses an 85 micron nozzle running at approximately 60Khz, (900,000 events/minute). We have found that this nozzle size works very well for most all the cells we sort – from lymphocytes to many cell lines. However, if you have a larger cell, or need a gentler sort, we can switch into our 130 micron nozzle, that runs at approximately 12 KHz, (180,000 events/minute). Switching from the 85 micron to 130 micron nozzle takes approximately 30 minutes, to allow for stream stabilization. Thus, if you are requesting a 130 micron sort, please build an extra 1/2 hour into your reservation to allow for this switchover.

We encourage investigators with new cells to provide a small sample of the cells a week before their proposed sorts. This will allow us to determine if the cells will sort efficiently on the 85 micron nozzle, or if the 130 micron nozzle will be necessary.

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.

Collection Tubes

Media in collection tubes should contain at least 10% serum. The appropriate volume of media for 5 ml or 15 ml collection tubes is 1 ml or 4 ml per tube, respectively.

It is important to pre-coat collection tubes with media + serum to increase post-sort viability and recovery. This involves coating the entire inner surface of the collection tube. Sorted cells that hit bare plastic have the potential to stick and dry out.

To aid in the recovery of a sort population of low percentage, a high volume of media in a conically shaped collection tube is recommended (13 ml in a 15 ml conical tube).