The MoFlo was purchased new from instrument grant money awarded to Bellur Prabhakar, Ph.D., head of the Department of Microbiology and Immunology in collaboration with the Research Resources Center. This instrument was installed in room E-20 of the Medical Sciences Building in the Spring of 2003. It was upgraded with a Smart Sampler the following December. It is a state-of-the-art instrument that can sort up to 50,000 cells/second under ideal conditions (this rate can be attained only with very concentrated, i.e. 40 million cells/ml) small cells that are not aggregated or sticky.

Extra features present on this instrument are:

1. Automatic cloning device called the CyClone. It can sort any given number of cells onto slides, or tissue culture plates (6 to 1536 wells).



2. 4-way sorting.



3. Temperature control.



4. The Sort Master, which monitors the sort and allows the operator to walk away after the sort has been started.



5. The Smart Sampler has adjustable tube holders, so the sample to be tested or sorted can be inside any size or type of tube.

Note: The MoFlo will be used mainly for sorting. Only experiments involving fluorochromes that can not be analyzed with our other flow cytometers will be scheduled for analysis on this instrument.

1. 5W water cooled Argon Ion Laser tuned to 488 nm: This common laser is used to excite Fluoroscein (FITC), Phycoerythrin (PE), PE-Cy5 (Cychrome/Tricolor), PE-Texas Red (ECD/Red 613), Red 670, Rhodamine 123, Propidium Iodide (PI), Acridine Orange, and other compounds. In addition, it can be tuned to other commonly used wavelengths such as 514 nm (TRIC) or 457 nm (NBD). Please inform RRC personnel if you need a non-488 nm wavelength to excite your fluorochrome.



2. 5W water cooled Argon Ion Laser tuned to MLUV. This laser is very good for exciting DNA specific dyes such as Hoechst and DAPI, and blue fluorescent protein e-BFP. Calcium flux can be measured using this laser. However, the UV wavelengths from the argon laser are a little too low to effectively excite Pacific Blue,Cascade Blue or eCFP.



3. 4W water cooled Krypton Ion Laser tuned for either violet wavelengths 406/413/415 nm or the yellow 568nm line. When tuned to the violet wavelengths, this laser can excite Pacific Blue, Pacific Orange, Cascade Blue, Alexa 430, and fluorescent protein eCFP. When tuned to the yellow 568nm line, this laser can excite TRITC, Alexa 568, DsRed and X-Rhodamine. When making an appointment for the MoFlo, always indicate the fluorochrome you will be using so that the Krypton laser will be equipped with the correct optics.
4. A 25mW 635 nm solid-state red diode laser: Used to excite Allophycocyanin (APC), Cy5 and the tandem conjugate APC-Cy7. This laser was added to the instrument recently; therefore we can now analyze/sort more multicolor combinations than before.

Helpful suggestions: To best utilize the high speed sorting ability of this instrument, the cells should be more concentrated than recommended for the Beckman Coulter Elite. We suggest giving us cells that are 10-30 million cells/ml. Sorting concentrations may be higher especially when the enrichment of rare cells/particles is desired. The cells should be placed in tubes in a minimum volume of 500 µl. Sorted cells can be deposited into either 12 x 75 mm or 17 x 100 mm round bottom centrifuge tubes. These tubes should be 75% full of medium or PBS containing high amounts of protein (i.e. fetal calf serum, bovine serum albumin, etc.).

For more details on what you need for sorting refer to our Sort Protocol page. Every time you sort you must fill out an analysis worksheet (PDF format).