The Moody Foundation Flow Cytometry Facility

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Introduction

Flow cytometry is a powerful technology for investigating many aspects of cell biology. Its power comes primarily from the fact that it quantitatively analyzes individual cells, thus permitting the identification of subpopulations of cells. The cell features in flow cytometry are detected primarily by fluorescence although significant information (size and granularity) is also contained in the way light is scattered by the cells. The power of single cell analysis is compounded by the ability to analyze multiple parameters simultaneously and by the ability (with appropriate instrumentation) to purify cell populations by high speed sorting.

Flow cytometers can analyze and sort cells at extremely high rates with the latest technology available in our facility. Some applications of flow cytometry include immunophenotyping (using fluorescently labeled antibodies or ligands), measurement of DNA and RNA content for cell cycle analysis, apoptosis, and cell viability. These techniques can be applied to a diverse range of experimental questions. An increasing application is the use of flow cytometry for preparation of gene libraries by sorting green fluorescent protein (GFP)-marked E. coli transfectants that are directly cloned following sorting. Although flow cytometry is mostly applied to the analysis of cells, it is applicable to any resolvable particle such as chromosomes or bacteria.

The use of flow cytometry is broadly divisible into two categories, analysis and sorting.

Analysis

Analysis is the interrogation of cells by a laser source and resolution of the available information from the probed cells. Analysis is used by all investigators who employ flow cytometry. Analysis can be performed from tens of cells/sec to thousands of cells/sec depending upon the instrument and sample.

Sorting

Sorting is the process of isolating identifiable subsets of cells. Sorting, of course, requires analysis to identify the cells of interest to be sorted. Sorting is done on a subset of flow cytometers equipped with the appropriate electronics. Cells are first interrogated and analyzed by the computer then those events meeting pre-selected criteria are sorted.

To sort, the cells are isolated into discrete, charged droplets, which are then deflected using a high voltage electrostatic field. Sorting generally results in highly pure populations, even when starting with very low frequency events (1-2 percent). We are able to sort at input rates upwards of 20,000 cells/sec. The speed of any particular sorting experiment is dependent on several factors. Up to four populations may be isolated simultaneously from a single sample labeled with up to 15 fluorochromes. The ability to simultaneously sort on several parameters gives flow cytometric sorting a unique capability.

The sorter can place defined numbers of cells into an array of collection devices including conical tubes, tissue culture plates of several different well configurations, and Tarasaki plates. This capability provides direct cloning technology useful in a number of applications including cloning of gene transfectants.