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- Configuration: Statler - Waldorf - Old Statler (Retired)
- Choosing a Sorter
- Sort Speed and Cell Size
- Proper Controls
- Adherent Cells
- Collection Tubes and Plates
Contact Matt: Email or 273-4020
Matt B, Sharleen, Cristy, and Terry can be reached at x6-5330.
Sort rates are: $35 setup fee and $85 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.
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:
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!
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.
What to bring with you for your sorting experiment:
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.
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.
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.
- Statler - 3-4128
- Waldorf - 3-4138