Flow Cytometry Core

Sorting of cells, nuclei, or similarly sized particles 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 software-based analysis, and apply a charge to deflect wanted cells or particles into a collection tube. Instruments include:

• 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.
• BD FACSDiscover S8: Based on input from DRC research base investigators, Joslin is planning to expand its offerings soon with a new instrument that brings on the capabilities for spectral flow sorting and image-based sorting. Spectral deconvolution will enable discrimination of more than 50 different cell features simultaneously and greatly improve the ability to distinguish signal from noise, with enhanced resolution, sensitivity, and compensation that significantly increase the fidelity of flow cytometry and sorting as compared to traditional flow cytometers. In addition, the ability to perform real-time image-enabled sorting, with coincident capture of detailed images of every cell as it passes the detector, will enable studies to quantify changes in cell morphology and spatial organization.

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.

The CTL ELISPOT reader is designed for scanning and evaluating a wide range of microtiter plate-based bioassays — in particular, ELISPOT assays. It is equipped with both brightfield and fluorescence optics, detecting up to 5 fluorescent channels. This instrument is also well suited for objective reproducible multiwell cell-counting of live cells.

Data analysis can be accomplished using a number of platforms, and the Core Manager consults with users to guide their analytic approaches. Recognizing that FlowJo software is the analysis package chosen by most users, the Core offers individual annual FlowJo user licenses at a substantially reduced cost.

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.