Flow Cytometry Core

Sorting of cells or nuclei is achieved through instruments that propel a suspension of particles one at a time through a narrow pressurized fluidics stream, interrogate by sample laser-based excitation of individual particles coupled to emission detection and computerized detectors, and apply a charge to deflect wanted particles into a collection tube. Instruments include:

• MoFlo Legacy: This cell sorter is capable of high speed and single-cell sorting on up to eight fluorescent parameters and is equipped with three lasers (488nm, 355nm, and 640nm). The instrument has the capability of four-way sorting into tubes or single-cell sorting directly into plates or slides.
• SORP BD FACSAria: This instrument is capable of high speed and single-cell sorting on up to 15 fluorescent parameters and is equipped with five lasers (355nm, 488nm, 405nm, 561nm, and 640nm). It is capable of four-way sorting into tubes or single-cell sorting directly into plates or slides. This instrument is equipped with an Aerosol Containment System for the sorting of BSL-2 biohazardous samples.
• BD FACSAria IIu: This self-run instrument is capable of high speed and single-cell sorting on up to 12 fluorescent parameters and is equipped with three lasers (488nm, 405nm, and 640nm). It is capable of four-way sorting into tubes or single-cell sorting directly into plates or slides. This instrument is also equipped with an Aerosol Containment System for the sorting of BSL-2 biohazardous samples.

These analyzers are based on FACS principles through a similar process as sorting but without separation of particles into separate collection tubes.

• BD LSR-II: This instrument is equipped with four laser lines (488nm, 405nm, 561nm, and 640nm) and 15 fluorescent detectors. The current configuration allows for two colors off the blue laser, five colors off the yellow-green laser, three colors off the red, and six off of the violet.
• Miltenyi Biotec MACSQuant: The MACSQuant is capable of detecting up to eight fluorescent parameters and is equipped with 488nm, 633nm, and 405nm solid-state lasers. This instrument has the ability to provide absolute cell counts.
• BD LSRFortessa: This instrument is equipped with five laser lines (445nm, 405nm, 488nm, 561nm, and 640nm) and 17 fluorescent detectors, and the HTS (High Throughput Sampler) system which enables fully automated sample acquisition from 96- and 384-well microtiter plates. The current configuration allows for two colors off the 445nm laser, three colors off the blue laser including GFP and YFP detection, five colors off the yellow-green laser, three colors off the red, and four off of the violet.

The Core maintains a Complex Object Parametric Analyzer and Sorter (COPAS) BioSort system that is specially designed to analyze and sort objects ranging from 40-250 microns.

An AutoMACS can be used to provide high yield enrichment of rare cell populations via magnetic selection, which helps to reduce per user sort times by enabling better pre-sort enrichment of target cells.

This application is supported by two pieces of equipment.

• A Cellometer Vision Trio Cellprofiler system combines bright field microscopy and fluorescence detection to generate both total cell count and fluorescence data at the bench. This system has been used for counting total cell numbers, measuring viability, and quantifying GFP expression.
• A Hemavet 950FS Multi Species Hematology System specializes in counting differential WBC with 24 parameters including total WBC, RBC, lymphocyte, monocyte, neutrophil and other granulocyte subtypes and platelet numbers. The system can be adjusted for mouse or human cell measurements.

This analyzer is designed for scanning and evaluating a wide range of microtiter plate-based bioassays—in particular, ELISPOT assays. This instrument is also well suited for objective reproducible multiwell cell-counting results.

For flow cytometry data analysis, the Core provides 3 FlowJo dongles that can be signed out from the core on a weekly basis.

An important function of the Flow Core is to provide expert training, not only for those users who are new to flow cytometry, but also for experienced users who wish to expand their research approaches and push the limits of the technology. It is recommended that potential users consult with Core personnel for fluorochrome combination feasibility and necessary controls prior to designing your analysis experiment.