Instrumentation

The Flow Sorting Facility houses two cell sorter instruments, FACSAria I and FACSAria II. The FACSAria I was upgraded in September 2010 and is equipped with 18 fluorescent PMT's and 405, 488, 561, and 640 nm lasers, and has bio-containment capabilities. The FACSAria II is equipped with four lasers (355 nm, 408 nm, 488 nm, and 633 nm) and has 13 fluorescent detectors. Both instruments have the capacity to do four-way sorts as well as sorting into microtiter plates (96-, 48-, 24-, 12-, or 6-wells) and onto slides. The instruments are operated by facility staff members and available during regular business hours by arrangement only.

Scheduling and Reservations

Sorts can only be scheduled by the sorter operators (Craig x1745, Kitty x8416). New investigators are asked to schedule an appointment to discuss their sorting needs with them. They can advise you on the best approach to get you the purity and yield that you require.
For each sort appointment, please fill out a Sort Request at the time the reservation is made. The complete information helps us understand the expected duration and instrument requirements of your sort (lasers, nozzle, and pressure to use). The Aria sorters can get booked well ahead of time, so please keep this in mind when scheduling.

At the start of each sort, we review the sort logic with you, to make sure that everything is set up how you want it. It usually takes about 15-30 minutes to pre-evaluate your sample and set up or adjust the instrument parameters. We typically sort at a rate of 5000-10,000 events/sec, or 18-36 million cells/hour. You can use these criteria to estimate how much time your sort will take.

Samples

Ideally samples are supplied at a concentration of 10-50x10e6 cells/ml. We will dilute if necessary, so please bring appropriate buffer if you need anything else but standard PBS for the dilution buffer. For small cell numbers, we suggest supplying the cells in at least 0.5 ml buffer or medium. Medium needs to contain less than 2% FBS (serum) and no phenol red. You can supply samples in any type of tube, but we will transfer them to 12x75mm style tubes prior to sorting.
To get an estimation of how many cells you need to bring, assume that you will get a yield of 50% of the targeted population. Our sorting efficiency is typically 75 – 90%, but there will be some loss due to cell death and other factors.

Samples must be filtered (<70 µm mesh) prior to sorting to prevent clogging of the instrument. We routinely use 35 µm nylon mesh cell strainers (BD #352235) for all samples that are not previously filtered or that require resuspension from a pellet prior to sorting. We may also re-filter samples when they start clumping during the sort. To prevent clumps in your sample due to release of DNA, it is recommended to add 10 U/ml DNase I. The collection tubes need to have a small amount of collection buffer or medium (~10% of the tube volume). This can be your growing medium or anything else that you would like to use. It is recommended to include FBS (serum) or BSA to coat the tubes with a cushioning layer prior to sorting.

Our Aria instruments are in a BSL2+ facility and special precautions are in place on the Aria I to run samples infected with specific pathogens that require such precautions. We ask investigators to discuss their specifics with us first.

SORT REQUEST FORM

Contacts

What to bring for
sorting experiments:

  1. Unstained cells – at least 250,000 in 0.5 ml, used for instrument setup.
  2. Single stained controls – for each fluorochrome used.
  3. The sample(s) to be sorted.
  4. Dilution buffer for these cells.
  5. Collection medium.