Experimental setup and sample preparation for the LSRII and FACSCaliburs

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

Self-Run PDF Guides

* FACSCalibur - Startup and shutdown.

* FACSCalibur1 - Changing the tanks, shutting down the HTS.

* FACSCalibur2 - Changing the tanks.

* LSRII - Startup and shutdown.

* LSRII - Changing the tanks.

* Basic analyzer troubleshooting.

* Connecting to your Home Drive.

Experimental Setup:

The last ten minutes of every appointment are spent bleaching and rinsing your cells out of the system. This cleaning step is essential to keep the machines running smoothly for all users. 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.

The FACSCalibur and LSRII are each able to collect information about a wide variety of fluorochromes. If you wish to run a fluorochrome not listed on the configuration please contact us prior to preparing samples. We may be able to run it on a different cytometer.

View optical configurations of the LSR II and FACSCaliburs.

Analyzer Sample Preparation:

Samples must be in a single cell suspension. The ideal sample concentration is dependent on the cell type you are sorting. As a starting point we recommend 1.0x10^6/mL. On the FACSCalibur, run no faster than 4,000 events/sec. On the LSR II, run no faster than 10,000 events/sec. Running faster greatly increases the chances of clogging the machine. We provide non-sterile saline to further dilute your samples if necessary.

Samples for the FACSCalibur or LSR II must be in the following tubes:

Analyzer Sample Filtration

If your samples clog the machine, you will be charged for any additional time necessary to remove the clog. We can not move other appointments to continue your appointment past the booked time. Therefore, it is imperative for your samples to be filtered immediately prior to analysis!

We require all samples to be filtered through a mesh no larger than 70um immediately before being placed on the machine. We highly recommend you filter here in the lab to reduce clumps caused by settling samples. Use of a larger mesh will leave aggregates that will clog the machine. Samples containing visible aggregates must be re-filtered 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.

Some filtration options:

We provide BD Falcon cell-strainer cap tubes (Falcon 352235) to users if necessary. However, if you need more than a few tubes supplied, we will ask you to replace them. An entire package of 25 tubes is $23.61.