UT, MD Anderson, Science Park - Research Division Fluorescence Activated Cell Sorting

The BD Biosciences FACSAria is a fluorescence activated (deflected droplet) cell sorter [FACS] with the capability of sorting cells utilizing four sort streams or in a single 96-well deposition mode. Single cell samples should be provided at a concentration of 5-30X10⁶ cells/ml*. Provide enough cells to comply with our standard operating procedure: 1) Initial cell count of sample to be sorted, 2) post sort analysis by flow cytometry, 3) post sort cell count. This will allow us to determine the purity of the sort as well as the recovery. Sterile sorting has additional requirements*. Usually a typical sort can provide greater than 98% purity. If a greater purity is required a pre-sort enrichment or re-sort is performed. A coincidence abort is employed in high purity sorting. Therefore one has the choice of purity or recovery, but not both.

* Notes:

In a sterile sort cell viability may be as important as purity. A high protein concentration may affect the sorting stability so it is recommended the sample tubes for sorting contain approx. 2% BSA in PBS or media with antibiotics. The receiving tube (12X75mm polypropylene Falcon # 2063) may be precoated with FBS and should contain 0.5ml 25% FBS/media (antibiotics - optional). Each receiving tube will hold approx. 400,000 cells (drops) with the 100um tip and 100,000 cells (drops) with the 130um tip. Please provide enough receiving tubes for collecting all the cells requested.

We utilize a 70um & 100um sort tip which is adequate for cells from 14um to 20um in diameter. Typically the orifice should be five times the cell diameter. The 130um tip is recommended for larger cells such as HeLa, keratinocytes and epithelial cells. Aggregates and debris will not only affect purity but sort speed as well. We adjust the rate of sorting by adjusting the sample cell concentration not by increasing the rate of flow. The rate of flow is related to the frequency developed to generate the droplets. For example the 70um tip uses 70psi and a frequency of approx. 90KHz. Ideallly one cell per 4 drops will ensure high purity with a low coincidence abort rate. Therefore one-fourth of 90Khz would translate into a sort rate of approx. 22,000 cells/second under ideal conditions. When cells are injected into a sheath fluid at 70psi the sample tube has an applied postive pressure of slightly less than 70psi creating a differential pressure and a core for the cells to flow within. The greaterer this differential the greater the integrity of the core and more stability to the flow stream. This stability is critical for stable and uniform droplet formation and a clean break-off point.

Adherent cell lines that become 'tacky' or 'stickey' in suspension pose problems as well. Some recommended solutions for keeping cells from forming aggregates or clumping is to resuspend single cell suspensions in Ca⁺⁺/Mg⁺⁺-free buffers, use EDTA up to approx. 5mM, use 2-5% PBS in lieu of FBS, addition of DNase II 10uL/mL, or the addition of Heparin 1 drop (10,000U)/sample tube as well as keeping the cell density below 5x10⁶ cells/ml.

High speed sorting (10,000 - 30,000 cells/sec.) employs a spcial high speed tip and greater sorting pressures (90 psi). An additional consideration is time. For example to sort 4X10⁶ CD4/8 double negative mouse thymus cells which comprise about 1-2% of the total CD4/8 cell population one would have to supply a minimum of 400X10⁶ cells. If the sorter sorts approx. 5,000cells/sec. this sort would take over 20 hours. A better strategy would be to increase the representative population for sorting by negative or positive cell selection, as performed with the Miltenyi MACS or Dynal magnetice bead-antibody system to deplete unwanted cells. This would reduce the sort time significantly as well as to increase the purity without affecting the cells sorted. By raising the 1% population to greater than 50% (easily achievable) 8X10⁶ cells would yield 4X10⁶ cells in less than 30 minutes.

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Last updated: 30-March-07