metastasis research protocols, vol2

Humana Press Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Metastasis Research Protocols Edited by Susan A. Brooks Udo Schumacher Volume II Analysis of Cell Behavior In Vitro and In Vivo Metastasis Research Protocols Edited by Susan A. Brooks Udo Schumacher Volume II Analysis of Cell Behavior In Vitro and In Vivo Cell Separations by Flow Cytometry 3 3 From: Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ 1 Cell Separations by Flow Cytometry Derek Davies 1. Introduction 1.1. Cell Analysis by Flow Cytometry Flow cytometry is a means of measuring the physical and chemical characteris- tics of particles in a fluid stream as they pass one by one past a sensing point. The modern flow cytometer consists of a light source, collection optics and detectors, and a computer to translate signals into data. In effect, a flow cytometer can be described as a large and powerful fluorescence microscope in which the light source is of a highly specific wavelength, generally produced by a laser, and the human observer is replaced by a series of optical filters and detectors that aim to make the instrument more objective and more quantitative. As a cell passes through the laser beam, light is scattered in all directions, and also at this point any fluorochromes present on the cell are excited and emit light of a higher wavelength. Scattered and emitted light is collected by two lenses—one set in front of the light source and one set at right angles to it. By a series of beam splitters, optical filters, and detectors the wavelengths of light specific for particular fluorochromes can be isolated and quantitated—up to six fluorochromes can be measured in some flow cytometers. A simplified diagram of the optical setup for two-color analysis is shown in Fig. 1. The theory of operation of flow cytometers is well documented, and there are several good general books on the subject (1–3). 1.2. Cell Sorting by Flow Cytometry Flow sorting may be defined as the process of physically separating particles of interest from other particles in the sample. Sorting can be accomplished by two flow cytometrically based methods: the electrostatic deflection of charged droplets (so-called “stream-in-air” sorters) (2,4) or mechanical sorting (5). Most 4 Davies commercially available cell sorters use the electrostatic method, which is based on the principles of droplet formation, charge, and deflection analogous to those used in ink-jet printers. Any fluid stream in the atmosphere will break up into droplets but this is not a stable process. However, by applying vibration at certain frequencies it is possible to stabilize the point at which droplets break off from the stream, the droplet size, and the distance between the drops. Therefore the time between the point at which the cell passes through the laser beam and is analyzed until its inclusion in a droplet as it breaks from the stream—the drop delay—is known and constant (Fig. 2). By calculating this time period, the drop- let containing the cell of interest can be specifically charged through the fluid stream the moment that the drop is forming. To avoid cell loss, the duration of the charging pulse can be altered to include either or both the preceding and the following drop. Charged droplets will then pass through an electrical field created by two plates—one charged positively, the other charged negatively. Droplets containing a charge will be attracted toward the plate of opposite charge and in this way will be separated from the stream. Fig. 1. Schematic of a simple four-parameter detection system: forward and right angle light scatter and two fluorescence parameters set to detect FITC (Fluorescence 1) and PE (Fluorescence 2) emission spectra. Cell Separations by Flow Cytometry 5 Mechanical sorters do not use the droplet method but rather employ a motor- driven syringe to aspirate the fluid containing the cell of interest (6). From a practical point of view mechanical sorters are relatively slow (maximum sort- ing speed of 500 cells/s) but they do have advantages: The system is enclosed, preventing both contamination and evaporation, and it is easier to set up and perform a sort and therefore a skilled operator is not a prerequisite. The sorting speed of stream-in-air flow cytometers varies depending on the manufacturer and the design of the machine from 5000 cells/s up to 20,000 cells/s. However, this is still relatively slow compared with bulk isolation methods such as cell filtration techniques or cell affinity techniques, as even at Fig. 2. Schematic diagram of a typical stream-in-air sorter. Cells are analyzed at the laser intersection (“moment of analysis”), enclosed in droplets at the breakoff point, and are charged at this point if they are to be sorted. High-voltage deflection plates attract cells of the opposite polarity. 6 Davies top speeds no more than 10 8 cells may be sorted in an hour. However in compari- son with other techniques flow sorting achieves the highest cell purity and recov- ery. In addition, such stream-in-air sorters are capable of sorting one, two, three, or four subsets which may be defined by quantitative and qualitative measure- ments of multiparametric cell characteristics, the number of which is limited only by the configuration of the flow sorter. It is also possible to adjust the mode of sorting depending on whether high purity (the default mode), high recovery (if a small, precious population is needed) or high count accuracy (for single cell sorting for cloning or polymerase chain reaction [PCR]) is required. 1.3. Applications of Flow Cytometry Anything that can be tagged with a fluorescent marker can be examined on a flow cytometer. This can be a structural part of the cell such as protein, DNA, RNA, an antigen (surface, cytoplasmic, or nuclear), or a specific cell function (apoptosis, ion levels, pH, membrane potential). As long as a specific cell popu- lation can be identified by its fluorescence characteristics it can be sorted. Examples of the applications of flow analysis and sorting are given in Table 1. The fluorochrome of choice will to a large extent depend on both the intended application and the illumination wavelengths available in the cytometer. The most common laser wavelengths and the fluorochromes that can be used with these are given in Table 2. The choice will depend on the number of cell characteristics being examined, as well as the spectral overlap between the fluoro- chromes and their commercial availability. The most common application of cell sorting is to separate a subpopulation of cells based on their specific phenotype, whether this be, for example, tumor cells from normal cells or cells expressing a particular antigen after transfection. To be able to successfully sort a subpopulation of cells, a sample must be in a single-cell suspension. This is generally achieved by enzymatic or mechani- cal dissociation. Once in a single-cell suspension, the cells of interest should be prepared by labeling with fluorochromes, to detect either antigenic determi- nants, structural components, or functional status that will allow them to be specifically identified. 2. Materials 1. Trypsin–versene: 0.02% EDTA (known as versene by many cell culturists; store at room temperature), 0.25% trypsin (store at –20°C). Add 4 mL of trypsin to 16 mL of versene. This mixture can be used for up to 1 wk if stored at 4°C. Warm the solution to 37°C before use. 2. Phosphate-buffered saline (PBS): 8 g of NaCl, 0.5 g of KCl, 1.43 g of Na 2 HPO 4 , 0.25g of KH 2 PO 4 . Dissolve in 1 L of distilled water. Check that the pH is 7.2. Autoclave for 20 min. Cell Separations by Flow Cytometry 7 3. Propidium iodide (PI; 50 µg/mL in PBS). This is light sensitive so should be stored in an opaque container at 4°C (see Note 1). 4. Trypan blue (0.4% w/v). Table 2 Common Fluorochromes Laser wavelength Examples 488 nm Fluorescein isothiocyanate (FITC) R-Phycoerythrin (PE) PerCP (peridinin chlorophyll protein) PE-Cy5 tandem conjugates, e.g., TriColor, Cychrome Propidium iodide Ethidium bromide Acridine orange Fluo-3 UV (ca. 350 nm) Aminomethyl coumarin DAPI (4,6-diamidino-2-phenylindole) Hoechst (33258 or 33342) Indo-1 Monochlorobimane 635 nm Allophycocyanin TO-PRO-3 Cy5 Table 1 Examples of Flow Analysis of Mammalian Cells Phenotyping (surface, cytoplasmic or nuclear antigen) (7,8) Cell cycle analysis (DNA or kinetics via bromodeoxyuridine) (9,10) Functionality, e.g., calcium flux, pH, membrane potential (11–13) Apoptosis and cell death (14,15) Enzyme activity (16,17) Monitoring drug uptake (18) Measurement of RNA or protein content (19) Fluorescence in situ hybridization (FISH) (20,21) Sterile sorting for reculture (2,6) Sorting of rare populations (22) Single cell sorting for cloning or PCR (23,24) Sorting for protein, RNA or DNA extraction (25) Chromosome sorting for production of chromosome-specific paints (26,27) Isolation of defined populations, e.g., tumor cells from normal cells (28) 8 Davies 5. Cell culture medium appropriate to the cell used, both with and without phenol red. 6. Fetal calf serum. 7. 70% Ethanol. Take 700 mL of absolute ethanol and add 300 mL of distilled water. 8. Nylon mesh: 35 µm and 70 µm (Lockertex, Warrington, Cheshire, UK; Small Parts Inc., Miami, FL). 3. Methods 3.1. Preparation of Cells for Flow Cytometry 3.1.1. Suspension Cells, for Example, Cultured or Primary Blood Cells 1. Perform a viable cell count using PI or trypan blue. Live cells will exclude the dye, whereas it will be taken up by cells whose membranes have been compromised. 2. Select the desired number of cells and decant into a sterile container. 3. Centrifuge at 800g. The length of centrifugation will depend on the volume of fluid. For small volumes (up to 100 mL), 10 min is sufficient; increase this for larger volumes (see Note 2). 4. Carefully pour off the supernatant, taking care not to disturb the pellet. 5. Resuspend the pellet in medium at a cells density of approx 10 6 cells/mL. 6. Perform antigen staining (see Subheading 3.2.). 3.1.2. Adherent Cell Lines or Primary Cultures 1. Remove culture medium by suction using a sterile pipet. 2. Add an appropriate amount of trypsin–versene (e.g., 10 mL per 10-cm dish, 20 mL per 250-mL flask). Wash the fluid around and discard all but a small volume (5 mL per flask, 2 mL per dish) (see Note 3). 3. Examine the cell monolayer microscopically at regular and frequent intervals and tap the vessel gently to aid the dispersion of cells. Incubate at 37°C if the progress is slow. 4. When the cell sheet is sufficiently dispersed, add an appropriate amount of growth medium with serum (e.g., 10 mL per dish, 20 mL per flask) and carefully resus- pend the cells in the medium. The addition of medium serves to neutralize the effect of the enzyme (see Note 4). 5. Perform a viable cell count using PI or trypan blue. Resuspend the pellet in medium at a cell density of approx 10 6 cells/mL (see Note 5). 6. Centrifuge at 800g for 10 min and again resuspend the pellet in medium at a cell density of approx 10 6 cells/mL. 7. Once cells are in suspension, antigen staining can be performed (see Subheading 3.2.). 3.1.3. Solid Tissue 1. Place tissue in a 10-cm sterile tissue culture plate and add 20 mL of enzyme (trypsin–versene) solution. 2. Leave for 15 min at 37°C, checking constantly for cell release. 3. If cell release is slow, tease gently using sterile forceps or a scalpel. Repeat this as necessary. Cell Separations by Flow Cytometry 9 4. Add medium containing 10% fetal calf serum to neutralize the enzymatic action. 5. Decant cells solution into a sterile 50-mL tube and centrifuge at 800g for 10 min. 6. Discard supernatant, perform a viable cell count, and resuspend cells at a density of approx 10 6 cells/mL. Repeat the centrifugation step. 7. Finally resuspend cells at a density of approx 10 6 cells/mL before antigen stain- ing (see Subheading 3.2.). 3.2. Antigen Staining 3.2.1. Directly Conjugated Antibody 1. Take cells at 10 6 /mL and centrifuge at 800g for 10 min. Carefully pour off the supernatant. 2. Add appropriate amount of fluorochrome-labeled antibody (see Note 6); incubate for 15 min at 37°C (see Notes 7 and 8). 3. Add medium to a cell density of 10 6 /mL. Centrifuge, discard supernatant and repeat. 4. Resuspend at 10 6 /mL in phenol red free medium containing a low level of serum or protein (no higher than 2%; see Note 9) in a sterile container before flow cytometry. 3.2.2. Indirect Staining of Antigen 1. Take cells at 10 6 /mL and centrifuge at 800g for 10 min. 2. Add appropriate amount of primary antibody (see Note 6); incubate for 15 min at 37°C (see Note 8). 3. Add medium to a cell density of 10 6 /mL. Centrifuge at 800g for 10 min, discard supernatant, and repeat. 4. Add fluorochrome-labeled secondary antibody at a dilution of between 1:10 and 1:20 (If the primary antibody is a monoclonal, this will generally be a rabbit antimouse antibody). Incubate for 15 min at 37°C. 5. Add medium to cell density of 10 6 /mL. Centrifuge, discard supernatant, and repeat. 6. Resuspend at 10 6 /mL in phenol red free medium containing a low level of serum or protein (no higher than 2%; see Note 9) in a sterile container before flow cytometry. 3.3. Preparation of the Flow Cytometer for Sterile Sorting 1. Sterilize the cytometer by passing 70% ethanol through all sheath and sample lines for 60 min. Wash out by replacing ethanol in the sheath container with sterile deionized water for 30 min, then replace this with sterile sheath fluid (PBS; see Notes 10 and 11). 2. Wash down all exposed surfaces—sample lines, nozzle holder, nozzle, deflec- tion plates, tube holders—with 70% ethanol. 3. Define cell population within the sample using the scatter characteristics of the particles in suspension (Fig. 3A) (see Notes 12 and 13). 4. Define the population to be sorted on the basis of fluorescence characteristics (see Fig. 3B,C). 5. Decide whether purity, recovery, or count accuracy is the most important factor and adjust the mode of sorting accordingly. Cells may be sorted into tubes; 96-, 24-, or 6-well plates- or directly onto slides (see Notes 14–18). 10 Davies Fig. 3. A typical three-color sort setup: cells have been stained with three fluoro- chrome labeled antibodies—FITC, PE, and TriColor. (A) Scatter characteristics of cells. A region (R1) is selected to exclude debris and include the single cell-population. (B) PE (y-axis) and TriColor (x-axis) fluorescence from this cell population. On the basis of these characteristics, a subpopulation is selected (R2) and the FITC fluorescence of these cells is shown in C. (D,E) These populations after sorting with the percentage purity. Cell Separations by Flow Cytometry 11 4. Notes 4.1. Preparation of Cells 1. Always wear gloves when handing potentially mutagenic chemicals such as propidium iodide. 2. When centrifuging it is advisable to avoid braking, as this can lead to cell loss. This also applies during subsequent antigen staining. 3. For some experiments, the use of trypsin may be contraindicated, for example, if the antigen under consideration is known to be cleaved by enzymatic action. In this case the cell sheet may be scraped from the vessel using a sterile cell scraper. This should be aseptic and only one cell scraper should be used for each vessel. 4. Phenol red in media may interfere with subsequent procedures, so it is generally advisable to use media without this when harvesting cells and subsequent anti- body staining. 5. If the cells still look clumped when examined microscopically, it is advisable to pass the cell suspension through a 21-gauge needle, which will help to disperse these but should have only a minimal effect on cell viability. 4.2. Antibody Staining 6. The amount of antibody added will depend on the number of cells to be stained and the concentration of antibody in the staining solution. This is best determined empirically in positive control cells by a pilot experiment of test dilutions to determine the optimal concentration. Always do these experiments on equivalent numbers of cells and remember to scale up the amount of antibody used when doing bulk staining. The dilutions required for optimal staining using commer- cial antibodies will vary widely. Also, in general, the dilution for flow cytometry is lower than for slide-based immunofluorescence, that is, a higher protein concentration is needed. Also it is important after washing steps to remove as much fluid as possible to avoid subsequent dilution of antibodies. 7. All pipets, tips, and containers should either be purchased sterile or should be autoclaved. 8. The length of time taken for antigen staining can vary—most antibody binding is very rapid (seconds), but some low-density, low-affinity antigens may take longer. The optimal temperature for staining is either 37°C unless using lymphocytes or other cells where antigen capping may occur in which case 4°C is preferable—in these cases the incubation time should be increased (doubled). 9. Immediately before flow sorting, cells should be suspended in low-serum or low- protein medium. Protein has a tendency to coat the sides of the sample lines in the cytometer and this can lead to blockages, which are best avoided. Collection medium, however, should contain serum and antibiotics. 4.3. Sorting 10. PBS is generally used as a sheath fluid, although any ionized fluid will be suit- able. Obviously this needs to be sterile for sterile sorting; sterility is achieved by [...]... basement From: Methods in Molecular Medicine, vol 58: Metastasis Research Protocols, Vol 2: Cell Behavior In Vitro and In Vivo Edited by: S A Brooks and U Schumacher © Humana Press Inc., Totowa, NJ 25 26 McWilliams and Collins membrane and extracellular components has contributed to the effects seen in the experimental and spontaneous models of metastasis described This view has been confirmed by in... no residual magnetism outside a magnetic field An iron-containing core is surrounded by a thin polymer shell to which biomolecules may be adsorbed From: Methods in Molecular Medicine, vol 58: Metastasis Research Protocols, Vol 2: Cell Behavior In Vitro and In Vivo Edited by: S A Brooks and U Schumacher © Humana Press Inc., Totowa, NJ 17 18 Clarke and Davies The beads can be coated in primary antibodies,... of other genes of interest in metastasis research The cell line used has been profiled for MMPs and possesses a wide range of the enzymes, but no invasive activity has been detected related to active MMP-2 (determined by substrate gel electrophoresis, zymography) By altering the levels of MT-MMP-1, we hoped to achieve an increase in active MMP-2 which has been linked to metastasis and therefore alter... Another assay for cell aggregation is coined “fast” (30 min) and allows numerical analysis (5) This assay is a modification of the technique described From: Methods in Molecular Medicine, vol 58: Metastasis Research Protocols, Vol 2: Cell Behavior In Vitro and In Vivo Edited by: S A Brooks and U Schumacher © Humana Press Inc., Totowa, NJ 33 34 Boterberg et al by Kadmon et al (7) For the preparation of a... available includes those coated in antibodies specific for human B (CD19) and T cells (CD2 and CD3) and T-cell subsets (CD4 and CD8), hematopoietic progenitor cells (CD34), and monocytes (CD14) For metastasis research two types of beads are available to separate tumor cells from blood or bone marrow For epithelial tumors, beads coated with antibodies against the human epithelial antigen are available... gelatinase A Int J Cancer 63, 568–575 5 Tsunezuka, Y., Kinoh, H., Takino, T., et al (1996) Expression of membrane-type matrix metalloproteinase (MT-1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay Cancer Res 56, 5678–5683 6 Alexander, C M and Werb, Z (1992) Targeted disruption of the tissue inhibitor of metalloproteinases gene increases the invasive behaviour of primitive... from highly purified CD14+ peripheral blood monocytes J Immunol 157, 3850–3859 Immunomagnetic Cell Separation 17 2 Immunomagnetic Cell Separation Catherine Clarke and Susan Davies 1 Introduction In metastasis research, it may sometimes be necessary to separate populations of tumor cells from a mixed cell population such as a tumor, peripheral blood, or bone marrow In addition, the normal counterparts... stability experiments and the freezing down of stock References 1 Stetler-Stevenson, W G., Aznavoorian, S., and Liotta, L A (1993) Tumor cell interactions with the extracellular matrix during imvasion and metastasis Annu Rev Cell Biol 9, 541–573 2 Parsons, S L., Watson, S A., Collins, H M., et al (1998) Gelatinase (MMP-2 and -9) expression in gastrointestinal malignancy Br J Cancer 78, 1495–1502 3 Bernhard,... Genetic Modification 25 3 Genetic Modification of Cell Lines to Enhance Their Metastatic Capability Daniel McWilliams and Hilary Collins 1 Introduction 1.1 Matrix Metalloproteinases and Transfection Metastasis is the final step in tumor progression from a benign cell to a fully malignant cell The metastatic phenotype results from a wide range of phenotypic changes in the cell from the expression of... in Chapter 19 by Haack et al in the companion volume The application of gene transfection experiments to produce clones of increased metastatic potential and their subsequent use in tumorogenesis and metastasis assays in nude mice are covered in Chapter 17 by Muschel and Hua, and the reader is directed to consult this chapter also 1.2 Definition of Transfection Transfected cell lines, known as transfectants, . U L A R M E D I C I N E TM Metastasis Research Protocols Edited by Susan A. Brooks Udo Schumacher Volume II Analysis of Cell Behavior In Vitro and In Vivo Metastasis Research Protocols Edited. Vivo Cell Separations by Flow Cytometry 3 3 From: Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo Edited by: S. A. Brooks and U. Schumacher. 3850–3859. Immunomagnetic Cell Separation 17 17 From: Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo Edited by: S. A. Brooks and U. Schumacher