Sorting Guidelines

The FACSVantage SE is a (BDIS) jet-in-air flow cytometer that enables cell sorting based on specific fluorescent parameters. Our instrument has both an Argon and a HeNe laser for excitation at 488nm and 633nm, respectively. A third laser (krypton) will soon be added to the optical bench allowing for UV excitation as well.  In addition to sorting into tubes, the CloneCyte Plus option enables sorting of specific number of cells into plates of varying well numbers or onto slides. Cell Quest software is utilized for both acquisition and analysis of list mode data files using FCS 2.0 format.  This instrument in analogue mode can collect cells from different populations into two different tubes simultaneously. In contrast, the instrument can be operated in full digital mode enabling four different populations to be sorted into 4 different collection tubes. 

The FACSAria is a new generation bench top flow cytometer/cell sorter and the first to incorporate a fixed alignment gel-coupled cuvette flow cell instead of the conventional jet-in-air configuration. The instrument can accommodate three solid state lasers with a variety of filters to enable detection of up to 13 different fluorescent color parameters as well as forward scatter and side scatter. This fully digital instrument samples the data 10 million times per second with each signal digitized into one of 16,384 possible levels. This eliminates the dead time inherent to analogue systems which are unable to process a new signal while in the midst of processing a previous signal. The FACSAria operates on a PC based Windows platform with the new FACS DIVA software and collects data in FCS 3.0 format.

Remember when planning your sort, that dead cells are sticky and can easily compromise the integrity of the sorted sample. Samples with viability less than 80% can result in lower purity of the sorted cells. Also, keep in mind that protein in your presorted sample can easily clog the sorter. In general, PBS without FBS is best, provided that your cells can survive without the addition of protein. Contact facility staff for additional information prior to sorting (410-955-7852).