Journal article Open Access
Author: Ingrid Schmid Corresponding author ([email@example.com](firstname.lastname@example.org)) ### INTRODUCTION Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G0 phase cannot be discriminated from cells in the proliferative G1 phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G0 and cell-cycle subcompartments of G1. Assessing phenotype at the same time as nucleic acid content allows determination of the cell-cycle status of subpopulations in mixed-cell preparations. This protocol describes an optimized method for combining dual-color cell-surface immunofluorescent staining with staining for DNA-RNA, adapted for a basic dual-laser flow cytometer with blue (488-nm) and red (633- or 647-nm) excitation. DNA is stained at low pH in the presence of saponin with 7-aminoactinomycin D (7-AAD), and RNA is stained with pyronin Y (PY). Both dyes are used at low concentration, and 7-AAD is exchanged with nonfluorescent actinomycin D to minimize fluorochrome-fluorochrome interactions, which can negatively affect detection of cell-surface antigen staining. ### RELATED INFORMATION This protocol was adapted from Schmid et al. (2000). An extensive description of flow cytometry principles, as well as various techniques for assessment of nucleic acid content by flow cytometry, can be found in Practical Flow Cytometry (Shapiro 2003). ### MATERIALS **Reagents** 1. Actinomycin D (AD) stock solution (1 mg/mL) - 7-aminoactinomycin D (7-AAD) stock solution (1 mg/mL) - Antibody staining solution - Human cells of interest, fresh or cultured (or immortalized human cell lines) - Monoclonal antibodies (mAb) directed against cell surface antigens of interest (biotinylated or labeled with an appropriate fluorochrome), and isotype-matched controls - *The protocol described here uses one biotinylated mAb in combination with streptavidin-Alexa Fluor 488 and one mAb directly conjugated to allophycocyanin (APC) for combining dual-color immunofluorescence with DNA-RNA staining. See Troubleshooting and Discussion for further information on strategies for fluorochrome selection and staining*. - Nucleic acid staining solution (NASS) (pH 4.8) - Phosphate-buffered saline (PBS, 1X, without Ca++ and Mg++) - Pyronin Y(G) (PY) stock solution (1 mg/mL) - Streptavidin-Alexa Fluor 488 ### Equipment 1. Bucket with ice - Centrifuge - Flow cytometer with 488-nm blue excitation and 633-nm or 647-nm red excitation, and the appropriate filter sets for collecting emissions from Alexa Fluor 488, PY, 7-AAD, and APC - Incubator, preset to optimal temperature for cells of interest ### METHOD **Cell-Surface Staining** 1. Place 1 × 10e6 PBS-washed cells into a tube, add 100 μL of antibody staining solution, and mix well. In addition, prepare cells to be stained with isotype-matched control antibodies, as well as cells to be stained with a single color, to be used as controls during flow cytometry. - *The preparation of samples stained with isotype-matched control antibodies is necessary for determination of background staining. The preparation of single-color control samples is necessary to determine the fluorescence overlap of APC and Alexa Fluor 488 into other detectors on the flow cytometer and to set accurate fluorescence compensation*. - Label the cell-surface proteins of interest: - i. Add appropriate amounts of a biotinylated mAb and an APC-labeled mAb to the cell sample. - ii. Incubate the cells protected from light, using the optimal incubation temperature and time for the antibodies selected. - *If the cell surface protein of interest is expressed at high levels, it may be possible to use a mAb that is directly conjugated to Alexa Fluor 488, omitting the need for indirect staining using a biotinylated antibody. If this is the case, omit Steps 3 and 4, and proceed directly to Step 5*. - Wash the cells once by adding 2 mL of antibody staining solution and centrifuging at 250g for 5 min at 4°C. Discard the supernatant. - To the cell pellet, add 100 μL of antibody staining solution containing an appropriate amount of streptavidin-Alexa Fluor 488. Incubate for 20 min at 4°C. - Wash the cells once by adding 2 mL of antibody staining solution and centrifuging at 250g for 5 min at 4°C. Discard the supernatant. **DNA and RNA Staining** 6.Stain cells with 7-AAD: - i. Resuspend the cells from Step 5 in 0.5 mL of NASS containing 10 μg/mL of 7-AAD. Incubate for 20 min at 20°C-25°C, protected from light. - ii. Prepare a cell sample that is stained only with 7-AAD (without any cell surface staining). - *This sample is necessary to determine the amount of fluorescence overlap of 7-AAD into other detectors on the flow cytometer and to set the appropriate fluorescence compensation*. 7.Add 1 mL of 1X PBS to each sample. Collect the cells by centrifugation at 250g for 5 min. Discard the PBS. 8.Stain cells with PY: - i. Resuspend each cell pellet in 0.5 mL of NASS containing 10 μg/mL of AD. Place the mixture on ice for 5 min, protected from light. - ii. Add 5 μL of a 1:10 dilution of the PY stock solution (made with H2O), and vortex immediately. Keep the cells on ice, protected from light, for at least 10 min before sample acquisition on the flow cytometer. - It is possible to keep cells in the staining solution protected from light at 4°C for a maximum of 3 d before sample acquisition on the flow cytometer, without adverse effects. - iii. Prepare a sample of cells stained only with PY (without any cell-surface or 7-AAD staining). - This sample is necessary to determine the amount of fluorescence overlap of PY into other detectors on the flow cytometer and to set the appropriate fluorescence compensation. 9.Run samples on the flow cytometer using a low sample differential. Collect the fluorescence signals for cell-surface immunofluorescence with log amplification. Collect the DNA and RNA signals with linear amplification (Fig. 1). ![Figure 1](http://i.imgur.com/UR8RC5I.jpg?1 "Figure 1") **Figure 1**. Application of the method for concurrent flow cytometric measurement of two cell-surface antigens and DNA-RNA content. Human peripheral blood mononuclear cells were cultured for 72 h, either in medium alone (A-D) or in medium containing 100 ng/mL of soluble CD28.2 (Biodesign) in the presence of 100 ng/mL of surface-bound OKT3 (Ortho Diagnostics) (E-H). (C and G) Analysis of CD5+CD8+ T cells; RNA/DNA dot-plots were gated on scatter as shown in row 1 (in A and E) and R1 (in B) or R2 (in F), respectively. (D and H) Analysis of CD5-CD8+ cells; RNA/DNA dot-plots were gated on scatter as shown in row 1 (in A and E) and on R3 (in B) or R4 (in F), respectively. After 3 d in culture without stimulation, CD8+ cells, irrespective of CD5 expression, did not leave G0. As expected, with CD3/CD28 activation, CD8+ cells that coexpressed CD5 (a human T lymphocyte antigen) proliferated, while CD5-CD8+ (NK cells) remained in G0. After 72 h of costimulation, only 9% of CD5+CD8+ T cells remained in G0; ~15% were in G1a, 21% were in G1b, and the rest were represented in the S + G2+M phases of the cell cycle. *Running samples at a low sample pressure lowers the coefficient of variation (CV) of the DNA measurement. Low CVs improve the accuracy of data analysis by facilitating deconvolution of DNA histograms by flow cytometry DNA analysis software programs. See Troubleshooting*. *A standard practice employed for reliable DNA analysis is doublet discrimination. This can be achieved by acquiring area and width signals in the channel used for measuring DNA fluorescence. If this is not possible due to instrument limitations, displaying area versus height signals or gating on light scatter signals (Fig. 1) can also be used to exclude cell aggregates during data analysis*. ### TROUBLESHOOTING 1. **Problem**: The DNA profiles have a high CV. **[Step 9]** - **Solution**: The low pH of the NASS improves access of 7-AAD to the DNA, resulting in DNA staining with low CV. Despite this, samples that are run through the flow cytometer at high sample pressure will be measured with decreased precision and higher CVs. In addition, there is fluorescence overlap between 7-AAD and PY. If the overlap is not compensated correctly, the 7-AAD peaks will widen. Also, if substantial numbers of cells are dying, their degrading DNA will lead to broad DNA distributions. Solutions involve removing dead cells by Ficoll gradient or choosing an earlier time point for analysis (before the cells start to die). - **Problem**: The detection of cellular subpopulations needs improvement. - **Solution**: This protocol was optimized for detection of surface antigens by using low nucleic acid dye concentrations and exchanging 7-AAD with nonfluorescent AD. This serves to reduce background fluorescence caused by the dyes in solution and to minimize fluorochrome interactions. Data obtained with these low concentrations matched the results obtained for DNA-RNA staining with standard concentrations of 7-AAD and PY. Therefore, because nucleic acid dye concentrations must be maintained at the levels described in the Method section, strategies to improve the brightness of signal detection should focus on the mAb staining step (Step 2). These strategies involve selecting the brighter fluorochrome for the cell-surface antigen with low abundance, titrating the mAb to achieve the best separation between background and positive cells, using indirect staining to amplify the fluorescent signal, and selecting different mAb clones that may provide better staining. ### DISCUSSION In mixed-cell preparations, analysis of surface antigen expression in combination with cell-cycle events is essential for the determination of the growth status of cellular subpopulations. Measurement of DNA content by flow cytometry using different fluorescent dyes for staining DNA is compatible with simultaneous detection of cell surface antigens, but only identifies the three basic phases of the cell cycle: G0/1, S, and G2+M. Cells that are actively progressing through the S phase can be identified in flow cytometry by combining measurement of DNA content with bromo-deoxyuridine (BrdU) incorporation, an assay comparable to the assessment of thymidine incorporation using a radioactive label. However, the DNA denaturation step required in many BrdU staining protocols to make incorporated BrdU accessible to anti-BrdU antibodies can affect simultaneous detection of other immunochemical parameters. Bivariate analysis of DNA content and cyclin expression, which varies during cell-cycle progression, provides a framework for the subdivision of the cell cycle into distinct subcompartments. The success of this technique relies on suitable cell-fixation, cell-permeabilization, and dependable antibodies specific for the various types of cyclin proteins that have been proven to work in flow cytometry. In addition to DNA content, the determination of RNA content is another approach for assigning cells to various cell-cycle compartments and for discriminating G0 from different stages within G1. Several techniques have been developed for DNA-RNA content measurement by flow cytometry. One method involves differential staining of DNA-RNA with acridine orange (AO) (Darzynkiewicz et al. 1980). Accurate measurement of DNA-RNA content using AO is rather complex and depends on stringent staining conditions. In addition, AO, due to its broad emission spectrum, cannot be combined with other common fluorochromes used for antibody labeling. Another method is the simultaneous analysis of DNA and RNA content using Hoechst 33342 and PY (Shapiro 1981). This assay requires ultraviolet excitation, which is not available on most benchtop flow cytometers. Alternatively, Toba et al. (1995) described a 7-AAD/PY DNA-RNA staining method that is compatible with cell-surface antigen staining with fluorescein-isothiocyanate (FITC)-labeled antibodies. This protocol describes a method optimized for the concurrent measurement of two cell-surface antigens using a standard dual-laser flow cytometer. Cell-surface staining is presented using one antibody that is directly labeled with APC, and a second, biotinylated antibody that is detected with Alexa Fluor 488-labeled streptavidin (Fig. 1). If a given cell-surface antigen is expressed at high levels, mAbs that are directly conjugated to Alexa Fluor 488 can be used, shortening the time required for staining. Conversely, it is possible to use one unlabeled mAb that is then detected with a fluorochrome-labeled species-specific antiserum. This strategy can be used to amplify the signal obtained with low-abundance cell-surface antigens. Note, however, that for dual cell-surface labeling, the first and second staining steps for the unlabeled mAb need to be completed before staining with the other, directly labeled mAb. Otherwise, the antiserum will bind to both mAbs (unless each was made in a different species). New fluorochromes have recently become available for flow cytometry that may be compatible with this protocol. The selection of fluorochrome labels for cell-surface staining with emissions that can be separated from the emissions of 7-AAD (peak emission at 650 nm) and PY (peak emission at 580 nm) is necessary. The fluorochromes must also be compatible with the acidic NASS solution (pH 4.8). For instance, fluorescein isothiocyanate (FITC) cannot be used, because FITC fluorescence is lost in acidic solutions. Before new labels are incorporated into this staining protocol, it is necessary to determine whether they retain sufficient brightness for detection of a given cell-surface antigen. ### ACKNOWLEDGMENTS The author thanks Dr. Steve Cole for performing cell cultures and the late Dr. Janis V. Giorgi for her support during the development of this method. National Institutes of Health awards CA-16042 and AI-28697 are acknowledged for grant support. ### REFERENCES 1. Darzynkiewicz Z., Sharpless T., Staiana-Coico L., Melamed M.R. (1980) [Subcompartments of the G1 phase of the cell cycle identified by flow cytometry.](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&journalCode;=pnas&resid;=77/11/6696) *Proc. Natl. Acad. Sci*. 77:6696–6699. - Schmid I., Cole S., Korin Y.D., Zack J.A., Giorgi J.V. (2000) [Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence](http://cshprotocols.cshlp.org/external-ref?access_num=10.1002/(SICI)1097-0320(20000201)39:2%3C108::AID-CYTO3%3E3.0.CO;2-4&link;_type=DOI). *Cytometry* 39:108–116. - Shapiro H.M. (1981) [Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1002/cyto.990020302&link;_type=DOI) *Cytometry* 2:143–150. - Shapiro H.M. (2003) *Practical Flow Cytometry* (Wiley Liss, New York), 4th ed. - Toba K., Winton E.F., Koike T., Shibata A. (1995) [Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1016/0022-1759(95)00050-K&link;_type=DOI) *J. Immunol. Methods* 182:193–207.