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The Children's National Research Institute

FACS Core Equipment

The CNMC FACS Core consists of four FACS analysis instruments located in Rooms 5340, 5220 and 5224, and one FACS sorter located in 5224. These include FACS Calibur DxP, CytoFLEX S, Amnis Imagestream X, Miltenyi MACSQuant and BD Influx. The Core also includes additional equipment necessary for the operation of the Influx cell sorter — a Gast air compressor with two gallon reserve to provide air pressure and a Nikon Axioskop fluorescent microscope for alignment of the lasers. A summary of each piece of equipment is included below.

BD Influx Cell Sorter

The Influx is a cell sorting instrument which is designed to rapidly separate viable cells from suspensions for sterile re-culturing, or further analysis and experimentation. The machine is designed for high purity enrichment of up to 98 percent purity, and can sort up to 12,000 cells per second. The instrument detects six fluorescent markers off of two lasers — FITC, PE, PE-Texas Red, PE-Cy5, APC and APC-Cy7. The Influx is also adaptable, and can sort individual cells directly into 96 well plates for single cell cloning, or any other standard sized plate, including 48, 24 or six well. Fluorescent expression data can be collected during the sort for later analysis. Users deliver samples for sorting by the trained Flow Core personnel.

Beckman Coulter CytoFLEX S

The CytoFLEX is an analyzer which utilizes avalanche photodiodes as an upgrade over traditional photon multiplier tubes found in other instruments to increase the sensitivity and linearity of the fluorescence detected. This instrument collects 13 colors off of four lasers, and can switch between single tube collection and 96 well plate collection. Users are trained for independent use on the machine.

Miltenyi MACS Quant

The MACS Quant combines magnetic aided cell sorting and flow cytometric analysis in a single instrument. Cells can be purified with the inline magnetic column using Miltenyi beads and then directly analyzed for expression of eight different channels — FITC, PE, PerCP-Cy5.5, PE-Cy7; APC, APC-Cy7; Pacific Blue and Am Cyan. The MACS Quant includes an automated sampler capable of collecting samples from 96 well plates. Startup and shutdown are automated for ease of use, and data can be analyzed on the instrument or exported for later analysis. Users can be trained for independent collection.

Cytek FACS Calibur DxP

This upgraded FACS Calibur is a cell analyzer which utilizes four lasers to compare expression levels of 13 colors and acquire events digitally. Events can be collected up to 10,000 events per second. The Calibur can be used for routine investigations of GFP fluorescence, DNA content and cell cycle analysis, and cytokine arrays using the BD Cytokine Bead array kits. The upgraded instrument collects five colors off of the blue 488 and yellow green 561 lasers (FITC, PE, PE-Texas Red/RFP, PerCP-Cy5.5, PE-Cy7), three colors off of the red 637 laser (APC, Alexa 700, APC-Cy7), and five colors off of the violet 407 laser (Pacific Blue, Am Cyan, eFluor 605, eFluor655, Qdot 705). The attached computer and software has been upgraded to acquire the digital data from the machine. Users can be trained for independent collection.

Millipore Imagestream X

This is a bench top analyzer which combines detailed cellular imaging with FACS analysis for robust statistical analysis of morphological changes within individual cells. The Imagestream X is equipped with two lasers (blue 488nm, 200 mW; red 642nm, 150 mW), and channels are designed for the collection of bright-field images (420-480), FITC (480-560), PE (560-595), PE-TxRd (595-642), PerCP Cy5.5 or APC (642-745) and APC Cy7 (745-800). Collection can occur via a 20x, 40x, or 60x lens objective. Pictures of every individual cell in all six channels are collected and stored for analysis of intracellular localization, differentiation and morphological events depending on experimental conditions. Data are collected on a Dell 8-core computer with 12 GB of RAM and are analyzed on a second Dell 8-core workstation with 12 GB of RAM.

All core equipment is maintained by Christopher Lazarski, and users are encouraged to ask questions about training, use and interpretations.