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Measurement of protein expression in live, intact cells using flow cytometry (FC) has been employed for several decades in the areas of immunology, cell biology, and molecular biology. More recently, this technique has found appreciation in applied scientific fields, including cancer biology and endocrinology, to serve as a tool for identifying cells more likely to respond to specific treatments. FC, also referred to as fluorescence-activated cell sorting (FACS), is an antibody-based method that provides the user with an ability to identify proteins expressed on surfaces of cells as well as in the cytoplasm, including steroid hormone receptors. This technique is most useful for examining specific cell types in a heterogeneous population and therefore can be used to identify cells more likely to respond to treatments based on expression of the appropriate receptor. Isolation of purified subpopulations for further manipulation and investigation of functional capacity is also possible using a cell sorter, which uses similar technology to isolate cells for use by the researcher. This is especially important for studying responses of less abundant cell populations in tissues that express high levels of a target protein or receptor of interest. Furthermore, FACS analysis is clinically useful to identify and isolate responsive cell populations, which may be less appreciable in whole tissues because of the diluting effects of surrounding, nonresponding cell types. Immune cells are commonly utilized as a source of cell populations in the FC technique and have previously been shown to express steroid hormone receptors and respond to steroid hormone treatment. Here, we demonstrate that FC is a useful tool for identifying immune cells expressing steroid hormone receptor protein. This method can also be easily expanded to include other, nonimmune cell populations to address specific research questions related to steroid hormone receptor biology.
Flow cytometry was first developed by researchers at Stanford University who set out to create a system for identifying and isolating live cells that could be used for further culturing and manipulation (1). This method creatively uses fluorescently-tagged antibodies to identify cells expressing a protein or receptor of interest (information on fluorochromes currently available for use is described and reviewed in ref. (2)). This method may also be similarly used with green fluorescent protein (GFP)-tagged antibodies. Samples are analyzed in a flow cytometer using lasers that can detect an array of information about cell. Detectors aimed directly in line with a single laser beam (forward scatter, FSC) determine its cell size while detectors aimed perpendicular to the laser beam (side scatter, SSC) assess granularity within the cytoplasm of cells (3–5). Fluorescent detectors within the flow cytometer are used to determine amount of fluorescence emitted by cells, which indicates level of protein expression. Currently, approximately 12 flourochromes can be analyzed at one time.
Flow cytometry is particularly valuable as it can determine not only whether a cell is expressing the protein of interest but also indicates the amount of protein expressed by a single cell on the basis of intensity of fluorescence. In addition, flow cytometry can be used to study expression of proteins on the surface of cells as well as those localized within the cytoplasm, which can be achieved using a simple permeabilization step. Given the powerful tool of flow cytometry, it is possible to determine specific cell populations within a tissue expressing such intracellular proteins as steroid hormone receptors to determine cells most relevant and more likely to respond to steroid hormone treatment. Previous studies by our group and others indicated usefulness of this technique in analyzing steroid hormone receptor expression in patient samples, cell lines, and murine models (6–10). Another advantage to this technique is that expression of proteins can be correlated with degree of activation, maturation, or differentiation of given cell types (11). Finally, if complex mixtures of cells are present, flow cytometry can be used to sort subpopulations of cells and identify steroid hormone receptors expressed by specific cell types for further investigation. We describe here a detailed method for assessing expression of steroid hormone receptor protein in immune cells collected from rats using flow cytometry and demonstrate the feasibility of looking at other cell populations using this method.
Live cells obtained from culture techniques or isolated from tissues can be easily prepared for analysis of protein expression using flow cytometry. Frozen tissues can also be considered for this method but should be allowed to grow overnight (at least 24 h) in tissue culture conditions to ensure nonviable cells are removed from the sample. Lymphoid organs, such as bone marrow and spleen, provide a plentiful source of immune cells. Other tissues, such as liver and kidney, can also be used for analysis of immune cells, although they have markedly reduced populations of immune cells. In addition, immune cells can be readily isolated from peripheral blood but require measures to remove erythrocytes in order to eliminate this unwanted cell population. The following includes detailed information on generating a single cell suspension to prepare for use in a flow cytometer. For cells that have been cultured, these can be collected and counted as dictated in Steps 3–6:
This work was supported by the Intramural Research Program of the National Institute of Mental Health (NIMH)/NIH and a biodefense grant from the National Institute of Allergy & Infectious Diseases (NIAID)/NIH Intramural Research Program.
1Charcoal-stripped serum (CSS) is used as some components of sera have demonstrated hormone-mimicking properties. CSS prevents unwanted changes in cellular activity that could potentially skew results of the experiment.
2Cell strainer may need to be moistened using 1× PBS prior to addition of sample to ensure even distribution and maximal filtration across unit.
3If cell collection results in high cell number yield, it will be important to dilute samples prior to obtaining cell counts. For this, a small portion (~50 µl) can be collected from a well-mixed sample and added in PBS to make the appropriate dilution (i.e., 1:20 dilution factor) prior to obtaining cell counts and determining cell viability.
4To ensure Ficoll layer is not disturbed, it might be useful to tilt 50-mL tube at an angle (approximately 30–45°) prior to adding sample. If Ficoll layer is disturbed during addition of sample, it will likely reduce viability as Ficoll is toxic to cells.
5To facilitate collection from tubes after Ficoll separation, it may be necessary to remove approximately 10–15 mL of supernatant (contains dead cells and tissue debris) before collecting leukocytes in cloudy region just above Ficoll layer.
6One of the major limitations of flow cytometry is antibodies recognizing a protein or receptor of interest must be available for use. Without an appropriate antibody that is specific to the protein, it is not possible to utilize this method to measure protein expression.
7If 1 × 106 cells/tube are not available due to the number of tubes required for appropriate analysis or a low cell yield, the researcher may use as few as 0.5 × 106 cells but no less than 0.25 × 106 cells per tube to ensure sufficient numbers of cells for analysis.
8Another common practice to loosen cell pellet prior to addition of antibodies is to gritch (scrape tube across grate of biosafety cabinet) instead of using a vortex.
9To permeabilize cells for intracellular staining, use of 0.3% saponin in 1× PBS can be substituted for the CytoFix/CytoPerm solution.
10For this protocol, only one steroid hormone receptor is analyzed for each tube. It is possible to analyze multiple steroid hormone receptors expressed by an individual cell but will likely result in problems with nonspecific binding due to multiple antibodies being introduced into the cytoplasmic region of cell. It is, therefore, highly recommended that the researcher analyze only one intracellular protein at a time in a single sample.
11It would be useful to include a sample that does not express the steroid hormone receptor of interest (serves as a negative control) and a sample that is known to express the steroid hormone protein of interest (serves as a positive control) for each experiment.
12There are a variety of flow cytometry instruments available from BD Biosciences, including FACScan, FACSAria, FACSCanto, and several other instruments that can be selected based on the needs of the researcher. In addition, there are other companies, including Beckman-Coulter, that offer a variety of flow cytometer instruments.
13In the event the computer does not recognize the flow cytometer, restart both the computer and flow cytometer in the appropriate order.
14If the fluorochrome is not stably coupled to the antibody, it will not be possible to identify fluorescence and measure amount of receptor expressed by cells. This could result in the generation of falsely negative data. Background fluorescence is sometimes higher in fixed cells, which occurs with use of the CytoFix/CytoPerm solution for intracellular staining of steroid hormone receptors. Therefore, titration of antibody to find optimal concentration for each sample may be necessary. It will be important to evaluate data generated from the flow cytometer and analyzed by software to gauge appropriate concentration for each experiment.
15FACSCalibur may require lines to be flushed if no events are being acquired by the flow cytometer. This may be due to the machine being clogged by clumps of cells or air bubbles that have formed. Other problems preventing the flow cytometer from acquiring events include a cracked sample tube (transfer cells to new tube); sheath reservoir too low or not properly capped (check fluid amount and placement of sheath cap); or waste reservoir being full (remove waste fluid).