Cell sorting is done on an Aria III (BD Biosciences) fluorescence activated cell sorter (FACS). Sorts can be optimized for purity, yield or single cell modes, into 2 or 4 tubes, or into tissue culture plates. The Aria III is equipped with blue (488 nm), green (561 nm), red (633 nm) and violet (405 nm) lasers with 11 detectors allowing for forward scatter (FSC), side scatter (SSC) and up to 10 colors.
Cell preparation is an important part of a successful cell sort. Viability and stickiness of cells must be addressed. We can give advice and point users to the literature however conditions may have to be determined empirically, and therefor more than one experiment should be expected. Please see our FACS SOP for some starter tips.
Please note that we are currently unable to sort or analyze un-fixed human or primate cells as the Aria III instrument is not in a BSL2+ facility.
The Aria III is calibrated each day of use with CS&T calibration beads (BD Biosciences) to ensure voltages and %rCVs are within acceptable ranges (e.g. bright bead %rCV <6%). For determining significance of a biological response, multiple controls and experimental samples must be assessed using standard statistical methods. Isotype controls, fluorescence minus-one (FMO) controls, and compensation controls should be used as deemed necessary by the Core facilitator. Rare events (<5%) are collected according to Poisson statistics to a desired %rCV using the equation CV=100/SQRT(#rare events).
Standard Operating Procedures
Have Questions? Contact Us.
Steve Orena, Lab Manager
Metabolic Molecular Phenotyping Core-Kannapolis Facilities
T: (704) 250-5041