Flow Cytometry Protocol

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Flow Cytometry in Cancer Research

Flow cytometry allows researchers to measure several physical characteristics of cells in suspension, such as cell shape, size, and internal complexity. By using a fluorescent compound to detect a specific cell component, the component can be further examined. For example, tumor cells often have abnormal amounts of DNA (aneupolid, i.e. having too many or too few chromosomes) compared to healthy diploid cells. This is the core application of flow cytometry in the area of Cancer Research.

The following functional assay is adapted from the book "Cancer Cell Culture: Methods and Protocols" ©2004 Humana Press, Product No. Z701424. This method is one of several detailed assays featured in this book. The book also contains detailed protocols on the isolation and culture of cancer cell lines.

Flow Cytometric DNA Analysis of Human Cancer Cell Lines

Peter Mullen, Author

From Methods in Molecular Medicine, vol. 88 Cancer Cell Culture: Methods and Protocols, pp. 248-255
S. P. Langon, Editor
Humana Press Inc., Totowa NJ, Copyright 2004

• Materials
• Methods
• Notes
• References

Materials

2.1 Preparation of Solutions

1. Citrate Storage Buffer:

   Add 85.5 g sucrose, S7903, (250 mM) and 11.76 g trisodium citrate dihydrate, C5920, 2 H₂O (40 mM) to a beaker of distilled water with 50 mLof dimethyl sulfoxide (DMSO), D2650. After adjusting to pH 7.6, fill to 1 L and store at 4 °C. This buffer is used/product/sial/81845 to store frozen samples prior to analysis ¹.

2. Stock Solution:

   Dissolve 2 g trisodium citrate dihydrate, C5920, (3.4 mM), 2 mLIgepal® CA-630, I3021, (0.1% v/v), 1044 mg spermine tetrahyrdrochloride, S2876, (1.5 mM) and 121 mg Sigma 7-9, T1378, (0.5 mM) in 2 L of distilled water after adjusting to pH 7.6. This "Stock Solution" is used to make up the digestion/staining buffers.²

3. Solution A:

   15 mg Trypsin, T0303, in 500 mLof Stock Solution (adjusted to pH 7.6).

4. Solution B:

   250 mg Trypsin inhibitor, T9253, and 50 mg RNase A, R4875, in 500 mLof Stock Solution (adjusted to pH 7.6).

5. Solution C:

   208 mg Propidium iodide, 81845, and 580 mg spermine tetrahydrochloride, S1141, made up in 500 mLof Stock Solution, adjusted to pH 7.6. Protect solution from the light by using metal foil throughout the preparation, storage, and the staining procedures (Note 2).

6. All solutions were stored in 20 mLaliquots at -70 °C.

2.2 Cell Culture

1. Select cell line of interest, for example, MCR-7 breast cancer³ or PE01 ovarian cancer⁴ cells.
2. RPMI/PS/10% FCS: Combine 500 mLRPMI 1640 culture media, R8758, with 5 mLpenicillin/streptomycin (PS), P4333, at final concentrations of 100 U/mLand 100 µg/mL, respectively. Add 55 mL(10 %) fetal calf serum, F6178 (Note 3).
3. Dulbecco’s phosphate buffered saline (PBS), D8662
4. 1X Trypsin-EDTA, T3924
5. 75-cm² and 150-cm² tissue culture flasks, (Corning^® Plasticware)
6. 10-mLsyringes and 21G needles
7. FACS tubes

2.3 Preparation of Samples for DNA analysis

1. Water bath and vortex mixer, Z258415
2. RNase, R4875

2.4 Acquisition of Flow Cytometric DNA Histograms

1. FACSCalibur™ flow cytometer (Becton Dickinson) (Note 4)

2.5 Analysis of Flow Cytometric DNA Histogram

1. Cell cycle analysis software, for example, ModFit LT™ (Verity Software House)

Methods

3.1 Cell Culture

1. Suspend 0.5 x 10⁶ MCF-7 breast cancer or PE01 ovarian cancer cells in 25 mLRPMI/PS/10% FCS and transfer to a 75-cm² tissue culture flask. Place in a 5% CO₂ humidified incubator at 37 °C.
2. Allow cells to grow to confluence, feeding twice per week by aspirating the spent media, washing with 20 mLPBS, and then adding 25 mLfresh tissue culture media.
3. When cells become confluent, remove spent tissue culture media and wash cells in 10 mLPBS. Remove excess PBS.
4. Add 5 mLTrypsin/EDTA to each flask and place back in the incubator until the cells have become detached (Note 5).
5. Transfer cell suspension to a sterile Universal container using a sterile pipet; pour 20 mLRPMI/PS/10% FCS (to deactivate the Trypsin) into the flask in order to wash it out and then pool with the cell suspension in the Universal container (total volume of 25 mL).
6. Centrifuge at 600 g for 5 minutes.
7. Pour off the media and resuspend the pellet in 5 mLRPMI/PS/10% FCS. Syringe with a 21G needle three times to break up the pellet, and then make up to 25 mLwith RPMI/PS/10% FCS
8. Count total number of cells using a hemocytometer.
9. Transfer aliquots of 1 x 10⁶ cells to FACS tubes (regardless of volume) and then centrifuge again at 600 g for 5 minutes.
10. Resuspend pellet in 100 µL citrate buffer, cover tubes, and store at –40 °C prior to analysis.

3.2 Preparation of Samples for Analysis

1. Thaw Solutions A and B, along with the frozen whole cell suspensions (in 100 µL) (but not heated) in a water bath at 37° before preparing and staining nuclei for DNA analysis. Allow Solution C to thaw at room temperature before placing on ice.
2. Digest cell suspensions down to nuclei by adding 450 µL of 0.003% Trypsin solution (Solution A), mix and leave at room temperature for 10 minutes (Notes 6 and 7).
3. Prevent further degradation by adding 0.05% (w/v) Trypsin inhibitor solution and 0.01% (w/v) RNase A (Solution B) in a final volume of 375 µL, mix, and leave for 10 minutes.
4. Finally, stain cells by adding 416 µL/mLice-cold propidium iodide/1.16 mg/mL spermine tetrahydrochloride solution (Solution C) in a final volume of 250 µL and leave the samples on ice in the dark for an additional 10 minutes prior to analysis.

3.3 Acquisition of Flow Cytometric DNA Histograms

1. This protocol assumes that the user is familiar with the principles and practices of flow cytometry and is able to run samples according to the operator’s manual pertaining to the instrument being used.
2. For the purpose of collecting data, all plots must be formatted for “Acquisition”.
3. Plot a two-parameter dot-plot of Forward Light Scatter (FLS) vs. Side Scatter (SS).
4. Plot a single-parameter FL2 (area) histogram with linear x-axis to illustrate relative DNA content (propidium iodide fluorescence is usually assigned to the FL2 channel; Note 8).
5. Plot a two-parameter dot-plot of FL2 (area) vs. FL2 (width) to monitor doublets (sub-heading 3.4).
6. Select the signal threshold (the point at which a signal will be accepted as a positive event) to FL2 and then set an appropriate value to gate out debris (a value of 20 should suffice in the first instance).
7. No compensation is required since only one fluorochrome (propidium iodide) is present.
8. Introduce the sample and set the machine to “Run”. Using the appropriate settings panel, adjust both FLS and SS photo multiplier tube (PMT) voltages so that the majority of dots in the first two-parameter dot-plot (FLS vs. SS) are contained roughly within the center of the box.
9. Adjust the FL2 PMT voltage up or down until the peak appears in the graph. The voltage can then be fine-tuned so that the main peak is approximately one quarter of the way along the x-axis within the linear graph. This voltage will allow sufficient space along the x-axis for the G₂/M peak (which will have twice as much DNA per cell) to be held in the graph and not over-spill the end. The histogram can also be monitored for any tetraploid cells that may be present.
10. After setting up the machine, 10,000 ungated events are collected. Data files are stored in an appropriate folder for subsequent retrieval/analysis using cell cycle software provided with the machine being used.

3.4 Analysis of Flow Cytometric DNA Histograms

1. Provisional analysis of data can be conducted in a manner similar to data acquisition with all histograms being formatted for “Analysis” rather than “Acquisition”.
2. Plot a two-parameter dot-plot of FL2 (area) vs. FL2 (width), open the first data file, and set the "gates" around the majority of cells contained within the FL2 (area) vs. FL2 (width) dot-plot. Define as "gate 1".
3. Plot a single-parameter FL2 (area) histogram with linear x-axis to represent relative DNA content. Format the histogram by deselecting the default "ungated" events and choose "gate 1".
4. Place cursors around the first (G₀/G₁), intermediate (S), and second (G₂/M) populations of cells in a manner appropriate to your machine and choose the appropriate statistics of interest. Successful analysis should yield the appropriate proportion of cells in the G₀/G₁-phase, S phase, and G₂/M phases of the cell cycle.
5. Accurate cell cycle analysis must be performed using dedicated software supplied with the instrument being used. Users are expected to be familiar with such software and the appropriate statistical models to use for such analysis. For the purposes of this protocol, analysis was carried out using the ModFit software provided with the FACSCalibur flow cytometer.
6. Open the ModFit program and select the appropriate FILE.
7. Choose the parameter for analysis; in this case select Fl2A for relative DNA content.
8. Define “gate 1” by selecting FL2A (x) and FL2W (y). Drag each of the points of the gate (R1) to include the entire cell population of interest.
9. Choose a specific MODEL to analyze the data or use the suggested model according to specified parameters, such as whether samples were fresh or frozen or paraffin embedded; of diploid, aneuploid or tetraploid DNA content; whether aggregates were present; or if there is a visible G₂/M fraction. The model can also account for the presence of internal standards should they be included.
10. Check the position and RANGE of the markers that are automatically placed on the histogram and adjust their position if necessary (this may be the case, particularly if the S-phase fraction becomes relatively high).
11. Calculate the relative cell cycle distributions using the FIT option. Repeat the process for all other samples, making sure that the cell population of interest is within the defined “gate”. Adjust the gate if necessary.
12. Data can then be tabulated and exported to a suitable presentation package, for example, Excel.

Notes

1. Many cancer cell lines are not true diploid but rather aneuploid, where aneuploidy is defined as having a greater than 10% deviation from the DNA index of 1.0. Many primary tumors can produce one or more aneuploid cell lines in addition to the normal diploid state. Established cancer cell lines usually exhibit a single diploid or aneuploid population.
2. Propidium iodide is a DNA intercalating agent and must therefore be treated appropriately and discarded in a suitable manner.
3. Fetal calf serum (FCS) must be heat-inactivated prior to use by heating in a water bath for 30 minutes at 56 °C. It can then be placed in 60-mLaliquots and stored at –20 °C.
4. Although much of this protocol pertains to a specific instrument, namely the Becton Dickinson FACSCalibur, the methodology is applicable to other machines. The manner in which histograms are set up and the cell cycle analysis software may be different.
5. The time interval for cells to become detached from the plastic is dependent upon the cell line being used, but is usually in the order of 5-10 minutes.
6. Chicken erythrocyte nuclei (CEN) and trout erythrocyte nuclei (TEN) can be added to samples prior to trypsinization and used as internal controls in order to calculate the ploidy (diploid DNA content of the cell line in question⁵. Because CEN and TEN have less total DNA per cell than human cells (35% and 80%, respectively), they have by definition DNA indexes of 0.35 and 0.8 relative to a human diploid index of 1.0. Relative positions (channel numbers) of the CEN or TEN peaks can therefore be used to determine the relative DNA index of unknown samples. Although both internal standards can be included when determining the ploidy value of a given cell line, cell cycle distribution is made difficult since the G₂/M peak of TEN will fall within the S-phase fraction of normal diploid cells. CEN alone can therefore be used if a ploidy check is deemed necessary while at the same time cell cycle data is required.
7.  There is considerable debate as to whether CEN and TEN controls are the most appropriate internal standards. A human blood preparation (with a true ploidy value of 1.0) may be more relevant, providing that the cell lines in question are not true diploid in nature (in which case the two peaks will overlap owing to their identical relative DNA contents).
8. All single-parameter histograms should be 1024 channels resolution (rather than 256) to obtain maximum resolution.

References

1. Vindeløv, L, Christensen, IJ, Keiding, N, Spang-Thomsen, M, Nissen, NI. (1983) . Long term storage of samples for flow cytometric DNA analysis. . Cytometry. 3, 317-322..

2. Vindeløv, L, Christensen, IJ, Nissen, NI. (1983) . A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. . Cytometry . 3, 323-327..

3. Soule, HD, Vazquez, J, Long, A, Albert, S, Brennan, MT. (1973) . A human cell line from a pleural effusion derived from a breast carcinoma. . J. Nat. Cancer Inst. . 51, 1409-1413..

4. Langdon, SP, Lawrie, SS, Hay, FGea. (1988) . Characterisation and properties of nine human ovarian cancer cell lines. . Cancer Res. . 48, 6166-6172..

5. Vindeløv, L, Christensen, IJ, Nissen, NI. (1983) . Standardisation of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and rout red blood cells as internal reference standards. . Cytometry . 3, 328-331..

Igepal is a registered trademark of GAF Corporation.
FACSCalibur is a trademark of Becton Dickinson.
ModFit LT is a trademark of Verity Software House, Inc.

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