Experimental setup and sample preparation for FACSAria Sorting

Biosafety

All users must complete and return a biosafety agreement form prior to running samples on any of our equipment equipment.

Our core is a multi-user facility where samples from various sources are analyzed and sorted. The safety of our staff and users is our utmost concern. Therefore, accurate information about sample sources, potential infectious agents, and sample preparation procedures is crucial for effective biosafety risk assessment. Failure to fully disclose important biosafety information may result in permanent denial of services.

Unfixed non-infectious human primary samples, other unfixed human samples such as cell lines, other unfixed non-human samples that carry material that could infect humans, or other unfixed samples classified as Biosafety Level 2 (BSL2) may only be run on the FACSAriaIIu in the biosafety cabinet. You must meet with us prior to running any such samples and provide us with appropriate documentation. All other human primary samples or samples which carry material that could infect humans must be fixed prior to running. You may not run any actively infectious or radioactive samples on any of our machines.

Biosafety Agreement Form

Experimental Setup:

The first ten minutes of every appointment are spent setting up the machine and optimizing settings. The last ten minutes of every appointment are spent bleaching and rinsing your cells out of the system. Please keep this in mind when determining how much time to book for your appointment.

It is imperative that you provide controls appropriate to your experiment. Most applications require:

  1. An unstained control of the same cell type as your samples: no stains, no antibodies, no viability dyes, no secondary antibodies.
  2. Single-stained controls of the exact same fluorochromes you are using in your experiment. Do not substitute different fluorochromes. (For example - FITC is not an acceptable control for GFP!)

We recommend you have single-stained controls that use the exact same antibody as your experiment samples. Use of other antibodies, especially when using tandem dyes, can result in incorrect sample settings. If you have any questions regarding controls, please contact us in advance so we may discuss your experiment.

You must be present during setting of gates for FACSAria sorting. Once setup is complete, you may leave as long as you provide us with contact information so we can reach you if there is a problem with your sort.

We do have other nozzle sizes and pressure options available on the FACSAriaII. Contact us prior to booking an appointment if you are interested in these alternative settings. Please keep in mind that changing machine pressures or nozzles require additional time to stabilize and set up the machine - approximately a half hour both before and after your appointment.

FACSAria Sample Preparation:

Samples must be in a single cell suspension. The ideal sample concentration is dependent on the cell type you are sorting. For mouse bone marrow, we recommend a concentration of 4x10^7/mL. For cultured cells, we recommend a concentration of 2x10^7/mL. Please bring spare sterile serum-free buffer and sterile filter pipet tips with you to dilute your samples further if necessary.

Samples for sorting must be in the following tubes:

FACSAria Sample Filtration

We require all samples to be filtered through a mesh no larger than 40um immediately before you bring them to be sorted. Use of a larger mesh will leave aggregates that will clog the machine. Samples containing visible aggregates must be refiltered prior to running on the machine. Sample diluent should not contain BSA or FCS at a concentration greater than 0.2%, as this can cause cells to aggregate.

If your samples clog the machine, you will be charged for any additional time necessary to remove the clog. It is imperative for your samples to be filtered immediately prior to sorting! We can not move other appointments to continue your sort past your appointment time. If you provide us with cells which will not stay in suspension we may not run them due to the increased chance of clogging the system.

Some filtration options:

We provide BD Falcon cell-strainer cap tubes (Falcon 352235) to users if necessary. If you need these supplied, you will be charged for an entire package of 25 tubes ($22.90).

Collection Tubes:

The FACSAria and FACSAriaII can sort up to four populations simultaneously.

All collection devices or tubes should be at least half-full with media. This media can contain BSA or FCS. We strongly recommend the addition of a general antibiotic to help prevent contamination. If you are sorting into tubes and would like your cells chilled we can set ice around the collection tubes to help keep them cool.

We highly recommend sorting small populations (less than 10,000 cells) into BioRad Titertubes. These 1mL polypropylene tubes are available sterile or non-sterile, with or without caps (biorad.com, cat no. 223-9390, 223-9391, 223-9395). These are suitable for simultaneous collection of up to four populations.

For larger populations, you can collect into BD Falcon 5mL tubes or eppendorf tubes. These are suitable for simultaneous collection of up to four populations.

For sorting many millions of cells, you can collect into 15mL conical tubes. These are suitable for simultaneous collection of one or two populations.

The FACSAria and FACSAriaII can also sort into plates. You can use Falcon 6-, 24-, 48-, 96-, or 384-well plates, Terasaki plates, and various other plate configurations. You can sort one cell into each well, or many cells per well.

If you would like to collect into a container not already mentioned please contact us prior to booking your appointment.