Sample Policy Sort Setup (Pressure Mode) Nozzle Size (µm) Applications High (70 psi) 70 High-speed sorting; robust cells Medium (35 psi) 70 Sorting of fragile cells, sensitive cells Low (20 psi) 100 Single-cell sorting, sorting of large or fragile cells; adherent cell lines Sample Policy SAMPLES MUST BE FILTERED through a maximum filter size of 40 µm The FACSAria™ uses a nozzle tip to generate a stream of sheath fluid. This nozzle has an orifice (70 µm or 100 µm) that when clogged results in the critical failure of the stream and most likely the sort as well (sample collection tubes can become compromised with sheath carrying non-target particles/cells as a result of the clog). As procedures and reagents used during sample preparation can induce clumping, such as centrifugation and high serum concentrations, filtering of samples should be the last step before they are sorted. Minimum Sample Volume: 0.5 ml (500 µl) The FACSAria™ can accommodate 1 ml, 5 ml, and 15 ml sample tubes. Samples should be resuspended in a standard PBS based FACS buffer w/ minimum serum (i.e. PBS + ≤ 1.0% serum). Sample Concentration Sort Setup Optimal Concentration (total cells/mL) High 40 x 106 Medium 20 x 106 Low 10 x 106 The system has several precision modes. The most commonly used mode is purity. In purity mode the system will always maintain purity at the cost of yield. Samples at high concentration, those above the optimal, will take less time to sort but will sort with a lower efficiency relative to those at optimal. Samples at low concentration, those below the optimal, will take longer to sort but will sort with a higher efficiency relative to those at optimal. Keep this in consideration when designing your sort experiment and scheduling time. Sample Collection Collection Devices 2-Way sorting into 1 ml, 5 ml, and 15 ml collection devices 4-Way sorting into 1 ml and 5 ml collection devices The FACSAria™ is equipped with a re-circulating water bath that allows specially designed 15 ml (2-way) and 5 ml (2-way/4-way) collection devices to be maintained at a set temperature point. The default set point is 4oC but can be modified to suit particular needs. Collection Tubes Media in collection tubes should contain at least 10% serum. The appropriate volume of media for 5 ml or 15 ml collection tubes is 1 ml or 4 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). Standard Sheath Fluid: HEPES Buffered Saline (10 mM HEPES, 137 mM NaCl) pH 7.4. Sheath fluid is prepared using Sigma-Aldrich Biotech. Performance Certified, Cell Culture Tested chemicals.