Flow Cytometry Facility

Flow Cytometry Facility
The Wistar Institute
3601 Spruce Street
Philadelphia, PA 19104-4268
Tel: 215-898-3811
Fax: 215-573-3786

Jeffrey S. Faust, M.B.A.
David Ambrose , B.S.
Daniel Hussey, B.S.

Introduction

Flow cytometry is a method, somewhat analogous to fluorescent microscopy, in which measurements are performed on particles (cells) in liquid suspension, which flow one at a time through a focused laser beam at rates up to several thousand particles per second. Light scattered and fluorescence emitted by the particles (cells) is collected, filtered, digitized and sent to a computer for analysis. Practically all of the work done at the Wistar flow cytometry facility involves measuring the binding of a fluorochrome-labeled probe to cells and the comparison of the resultant fluorescence to the background fluorescence of unstained cells. See the following discussion for examples of the types of probes we can analyze and the types of assays we can perform.

The sister discipline of flow cytometry is cell sorting, in which the particles (cells) are separated and recovered from suspension based upon properties measured in flow.  Cells that are recovered via flow sorting are viable and can be collected under sterile conditions. We can most often recover subpopulations that are in excess of 99% pure.

(For schematic views of a flow cytometer FRONT, please click here.)
(For schematic views of a flow cytometer OVERHEAD, please click here.)

FACS is an acronym, and brand name, for Fluorescence-Activated Cell Sorting. This term is used often, and inaccurately, to describe standard flow cytometry analysis.

What is measured with flow cytometry?
Here at The Wistar Institute, practically all flow cytometry is used for measuring parameters of cells. Parameters include physical and/or chemical characteristics associated with cells or properties of cell-associated reagents or probes, any of which are measured by instrument sensors. (Click here for a brief (and incomplete) list of parameters.)

Common flow cytometry applications
The following are examples of measurements utilized in routine experiments using flow cytometry.

Light Scatter
Forward and side scatter are used for preliminary identification and gating of cell populations. Scatter parameters are used to exclude debris, dead cells, and unwanted aggregates. In a peripheral blood or bone marrow sample, lymphocyte, monocyte and granulocyte populations can be defined, and separately gated and analyzed, on the basis of forward and side scatter.

Fluorescence
Fluorescent parameter measurement allows for investigation of cell structures and functions based upon direct staining, reactions with fluorochrome labeled probes (e.g., antibodies), or expression of fluorescent proteins. Fluorescence signals may be measured as single or multiple parameters corresponding to different laser excitation and fluorescence emission wavelengths.

When different fluorochromes are used simultaneously, signal spillover can occur between fluorescence channels. This needs to be corrected through compensation. Certain combinations of fluorochromes are not able to be used simultaneously – please consult with someone experienced in flow cytometry prior to planning multi-fluorochrome experiments.

Immunofluorescence involves the staining of cells with antibodies conjugated to fluorescent dyes such as FITC (fluorescein), PE (phycoerythrin), APC (allophycocyanin), and PE-based tandem conjugates (R670, CyChrome, etc.). Cell surface antigens are usual targets of this assay, but antibodies can be directed at antigens or cytokines in the cytoplasm as well.

DNA staining is used primarily for cell cycle profiling, or as one method for measuring apoptosis. Propidium iodide (PI), the most commonly used DNA stain, cannot enter live cells and can therefore be used for viability assays. For cell cycle or apoptosis assays using PI, cells must first be fixed in order for staining to take place (see protocol). The relative quantity of PI-DNA staining corresponds to the proportion of cells in G0/G1, S, and G2/M phases, with lesser amounts of staining indicating apoptotic/necrotic cells. PI staining can be performed simultaneously with certain fluorochromes, such as FITC and GFP, in assays to further characterize apoptosis or gene expression.

Gene Expression and Transfection can be measured indirectly by using a reporter gene in the construct. Green Fluorescent Protein-type constructs (EGFP, etc.) or ß-galactosidase can be used to quantify populations of those cells expressing the gene/construct. Mutants of GFP are now available which may be excited at common frequencies, but emit fluorescence at different wavelengths. This allows for measurement of co-transfection, as well as simultaneous detection of gene and antibody expression. Appropriate negative (background) controls for experiments involving GFP-type constructs should be included – the control is the same cell type, using the gene insert minus the GFP-type construct.

Metabolic Studies and other
Annexin-V may be labeled with various fluorochromes in order to identify cells in early stages of apoptosis.

CFSE binds to cell membranes and is equally distributed when cells divide. The number of divisions cells undergo in a period of time can then be counted. CFSE can be used in conjunction with certain fluorochromes for immunofluorescence.

Calcium flux can be measured using Indo-1 markers. This can be combined with immunofluorescent staining.

Intercellular conjugation assays can be performed using combinations of dyes such as calcein or hydroethidine.

Cell Sorting refers to an assay in which cells are separated and recovered from suspension based upon properties measured in flow cytometry analysis.  Up to 4 subpopulations may be separated by instruments at Wistar.  Most assays utilized for analysis may serve as the basis for sorting experiments, as long as gates and regions defining the subpopulation(s) to be sorted do not logically overlap.  Throughput rate maxima range from 10,000 cells/second (36 x 106 cells/hour) to 25,000 cells/second (90 x 106 cells/hour).  Throughput rate is dependent upon cell size; smaller cells, such as lymphocytes, may be sorted with a smaller nozzle (70μ) at the higher rates, whereas larger cells such as most cultured cell lines are sorted with the larger nozzle (100 μ) at eh lower rates.  The rate of collection of the separated population(s) sorted depends primarily upon the condition of the cells and the percentage of reactivity.  Cells that are recovered via flow sorting are viable and can be collected under sterile conditions. We can most often recover subpopulations that are in excess of 99% pure.  (Click here for a guide to cell sorting.)
Common cell sorting experiments usually involve immunofluorescence assays - staining of cells with antibodies conjugated to fluorescent dyes in order to detect antigens.  (See discussion of immunofluorescence).  In addition, a great deal of sorting is done using GFP-type constructs in order to isolate pure populations of cells expressing a given gene/construct.  (See discussion of gene expression).  Other criteria may also be utilized for cell sorting – please check with the facility.