Cytometry

Experimental Design

Successful flow cytometric experiments require careful design to take into account the expected levels of antigen expression, spectral overlap of fluorochromes used to detect cellular antigens, the limits of antigen detectability, the number of cells to be measured and the inclusion of appropriate controls.  The Cytometry Center offers consultation in the design of experiments and in the selection of appropriate antibody combinations.

Antibodies

Relatively simple flow cytometric experiments can require 10 to 20 different antibodies.  The smallest quantities of labeled antibodies typically cost from $100 to $300 such that a single experiment can cost thousands of dollars.  As the result of initial BRIN/INBRE support and continuing recharge fees, the Cytometry Center has acquired an extensive panel of fluorescent monoclonal antibodies that have been titrated and characterized.  These antibodies are available on a cost recovery basis so that an experiment requiring 10-20 antibodies would typically cost less than $100.

Data Acquisition

The Cytometry Center has developed acquisition protocols for many types of cells and many different combinations of antibodies.  Data can be acquired using these existing protocols, modified versions or with completely new protocols to ensure that adequate numbers of cells are analyzed.   All data files are archived by the Cytometry Center on multiple computers and on optical media for long term storage.  Copies of data files can be sent to other laboratories over the UNR network.

Data Analysis

Properly designed and executed flow cytometry experiments require appropriate analysis.  Although flow cytometric data are collected and stored in standardized formats, general purpose software such as spreadsheets cannot import or analyze these files.  Specialized software is required and the Cytometry Center has FlowJo from Tree Star Software which is one of the leading programs for analyzing flow cytometric data.   A PowerPC Mac computer with FlowJo is available for use in the Cytometry Center for analyzing data.  FlowJo includes the capability to export graphical and tabular results to other software for inclusion in presentations and manuscripts.  The computer in the Cytometry Center has Adobe Illustrator for creating figures, Adobe InDesign for creating posters and complex documents, Apple iWork software for documents and presentations and Microsoft Office.

Cell Sorting

Although not formally a part of the INBRE UNR Cytometry Center, the Beckman Coulter Elite flow cytometer and cell sorter maintained and operated by the Department of Physiology and Cell Biology is available for use by UNR laboratories.  The Elite is an intermediate speed sorter capable of operating at rates up to ~12,000 cells per second.   The Elite can perform two-way bulk sorts for cells to be cultured or examined in other ways including expression microarray (“gene chip”) analyses.  In addition, the Elite has AutoClone capability that can be used to distribute one or more cells per well in tissue culture plates of several sizes, e.g., 96, 48, 24, 12 or 6 wells per plate.  This feature is extremely useful for cloning cell lines and has been used with stem cells, hybridomas and several tumor lines.

The Elite is a typical stream-in-air droplet sorter that creates aerosols during its operation.  Since it is not housed in a negative pressure facility, human materials and samples with infectious agents cannot be sorted.