Please follow these guidelines for cell sorting:

1. Sorting appointments can be made for sorting cells that are “known” to the flow lab. If you have a history of running a particular type analytical experiment in the core lab, then it may be possible to jump to preparative work without making a “pre-sorting” appointment for a small scale analytical experiment(s) first. However, if the application you are using has not been worked out on a flow cytometer in this core lab, please do not expect to jump into a large preparative experiment on your first visit.

2. Be sure to have some 5 ml round bottom tubes with strainer caps available (Falcon Tube #2235). Alternatively, you can run your cells them through another kind of sieve from Falcon. The 70 micron sieve cat. # 352350 works well, especially for larger volumes.

3. It cannot be overemphasized how detrimental extensive cell-cell adherence is to the success of your sort. For people trying to sort adherent cells, this problem often overrides all other difficulties, and the quality of most of our sorts of adherent cells, is proportional to the success of the cell dissociation methods used on the sample.

   The purpose behind a cell sorter’s design is to separate particles by their individual properties. For obvious reasons it is not possible for the instrument to separate cells by their individual properties if they are not a suspension of individual cells. Under conditions resulting in aggregate formation, the material sorted may be no more purified than it was in the beginning, and it may also clog the instrument.

   The reason that the importance of this critical step is often underestimated is because most cell harvesting is not done with flow cytometry in mind. When harvesting cells only for purposes of subculture, one only needs to make a successful transfer from one flask to another. This will work even with partially aggregated cells. However, that kind of harvesting is not sufficient preparation of cells for sorting. Harvesting for flow cytometry and cell sorting is much more demanding and will likely require a change in your procedures if you have not successfully sorted your cells before. The cells must be dispersed into single cell suspension (see below). If they are damaged and leaking DNA from harvesting, then the presence of loose, viscous DNA will persist even after sieve-filtration, and continue to trap cells into clusters. This is not uncommon. An aggregated cell preparation is of limited usefulness for sorting, and will likely ruin all your work with respect to the sorting effort.

   To solve these harvesting problems we recommend, instead of Trypsin or scrapping, the use of other enzyme mixtures for harvesting. Accumax® from Chemicon or Liberase® from Roche may be useful. Accutase or Accumax from CHEMICON International, Inc – [28820 Single Oak Drive - Temecula, CA 92590 - Ph: 951-676-8080: Accumax cat. # SCR006 / 100ml / $24]. It is a gentle mixture of enzymes (including DNase in the Accumax). Also, cells must be filtered just prior to sorting! Counting cells is best done after they are filtered. Samples with aggregates can easily clog a 70 uM nozzle and delay sorting.

4. Prepare your cells as you normally would for flow cytometry analysis. Cells to be sorted may be in a 5 ml round bottom tube or 15 ml conical tube in buffer with a small amount of protein. It is highly recommended that you count your cells immediately prior to sorting. This will allow an estimate of efficiency and the ability to trace problems associated with low yield. The cell concentration should be approximately 10-20 million cells/ml. If you only intend to clone cells, then the concentration can be one tenth of this. If you have fewer cells, a lower concentration is acceptable; however sorting time is extended as the cell concentration decreases.

5. When you present the cells to be sorted, you must supply 5 ml round bottom tubes or 15 ml conical tubes. These tubes should contain 1 ml media or buffer with protein. Keep in mind that the collection medium will be diluted with PBS as the cells are sorted into the collection tubes.

   Sorting into other containers such as 96 well plates is also possible with a much smaller "cushion" of media. Please note!!! Before making the appointment for the work, you must inform the flow lab manager what kind of vessel will be used to receive the sorted cells.

6. A meaningful negative control is necessary for background determination. Also single positive controls for each fluorochrome used in the experiment should be provided to calculate compensation.

7. Please keep your cells on ice, until loaded onto the sorter.

The UTMB Flow Cytometry Core Facility in the Department of Microbiology and Immunology is prepared to aid and advise investigators in their experimental design, instrumentation, data analysis, and assay development. The facility is located in the Medical Research Building room 3.159. It is directed by Dr. Janice Endsley, an immunologist and associate professor in the Department. Its daily operation is managed by Mark Griffin. Mark is has been working in flow cytometry and cell sorting for 26 years, and has been with UTMB for 20 years.

The facility is equipped with one high-speed cell sorter as well as an analytical flow cytometer. ANALYSIS: The BD LSRFortessa is an advanced analyzer that can detect 18 colors of fluorescence. The Fortessa offers automatic sample loading with a 96 well plate reader that allows for walk-away use. CELL SORTING: The cell sorting service is available through our BD FACSAria IIU, an operator-assisted sorter that can separate cells into tubes, plates, or slides at high speed. Direct cloning of sorted cells into tissue culture plates is also possible under aseptic conditions. In addition to its cell sorting functionality, it is capable of 9-color analysis in its present configuration.

The flow lab can also provide assistance with protocol development and implementation, as well as data analysis and presentation. The data from the Aria and Fortessa are available campus-wide over the university's intranet for one month, which allows investigators analyze and store data in their own labs. For scheduling work, researchers can email mgriffin@utmb.edu. The operator’s assistance is available 9-5 daily. A schedule with instrument availability is kept here on the Flowcore website. Call (409) 747-2011 for general or operational issues.

For all other inquiries and comments, please contact Meredith Weglarz.

FloJo site licenses are available at UTMB and are currently administered by Mark Griffin.

Instructions for obtaining a license can be viewed here.