Sorting

Fee Structure:

Sort rates are: $30 setup fee and $80 an hour billing, with a 1 hour minimum. Time is then billed to the minute. 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.

Sorting Guidelines

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:

• You must fill out a sort form or we cannot sort.
• When scheduling time, please plan on an extra 1/2 hour for instrument setup. The sorter is sterilized every day as part of QC.
• When sorting yeast, please book an extra 1/2 hour for instrument clean-up as part of your sort time.
• For investigators who need to use the 100 or 130 micron nozzle, please reserve an extra 1/2 hour to allow for nozzle changeover. You will also need to reserve the large nozzle on the calendar.
• Investigators that need to sort within 48 hours of a block of time must call the Flow Core and speak with Matt Cochran or Tim Bushnell. A voicemail is not sufficient.
• 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, 5 ml, and 15 ml tubes.
• Samples should be at 30x10e6 cells/ml for standard nozzle (85um), and 20x10e6/ml for 100um or 10x10e6/ml for 130um sorts.
• Reservations now have a place to designate nozzle size for your sort. Please fill in the nozzle size you need or we will assume it is a standard sort with the 85um nozzle.

Choosing a Sorter

• If you are doing a GFP/YFP sort, you must use Statler.
• If your sort uses Qdots, you must use Waldorf.
• If your sort uses mCherry, you must use Waldorf.
• If your sort uses PerCP, you must use Statler.

If you have a question based on these restrictions or have concerns about your sort, please contact a staff member so we can discuss your needs!

Sort Speed and Cell Size

The default configuration of the FACSAria uses an 85 micron nozzle running at approximately 60Khz, (36 million events/hour). 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 100 micron nozzle, that runs at approximately 25 KHz, (20 million events/hour). Switching from the 85 micron to 100 micron nozzle takes approximately 30 minutes, to allow for stream stabilization. Thus, if you are requesting a 100 or 130 micron sort, please build an extra 1/2 hour into your reservation to allow for this switch over.

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.

Proper Controls

What to bring with you for your sorting experiment:

1. Unstained Cells – so we can establish the size and fluorescent background of your cells.
2. Single Stained Controls – for each fluorochrome you are using.  These can be either beads ( “Simply Cellular Compensation Standard” beads from Bangs Lab, “BD CompBeads” from BD Biosciences, or “AbC beads” from Life Technologies) or cells. It is important that you have a positive and negative in your control samples.  Likewise, if you use beads for compensation, please make sure to provide a negative (unstained) bead as well.  
3. Your cells at the appropriate concentration
4. Sufficient catch tubes for your populations.  

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 and Plates

Media in collection tubes should contain at least 10% serum. The appropriate volume of media for 5 ml or 15 ml collection tubes is 0.5 mL or 1.5 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).

Collection Tubes can hold the following number of cells based on the nozzle used. These numbers are optimal. This means that collection tubes have 1/10 the volume as collection media (ie, a 5mL tube will have 0.5mL of media in it).

For sorting, various size plates can also be used for collection. Those plates are all BD Falcon Polystyrene plates with 6, 24, 48, 96, or 384 wells. Sorting onto glass slides, frosted or unfrosted, is also possible.