a Beckman Coulter Life Sciences: White Paper

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1 a Beckman Coulter Life Sciences: White Paper Violet-Excited nim-da Allows Efficient and Reproducible Cell Cycle Analysis on the Gallios Flow Cytometer Authors: Valdez, Ben 1. Carr, Karen 2. Norman, John 2. Affiliation: 1 Department of Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Beckman Coulter, Inc. Miami, FL, USA

2 Violet-Excited nim-da Allows Efficient and Reproducible Cell Cycle Analysis on the Gallios Flow Cytometer BACKGROUND Analysis of cell replication phases can be achieved by labeling cells in suspension with a DNA-specific fluorescent dye and analyzing the DNA content of individual cell with flow cytometry. Otherwise known as cell cycle analysis, this assay has been an important and consistent flow cytometry methodology since its advent in 1969 [1, 2], and applications of cell cycle analysis in the study of drug cytotoxicity and genetic modifications have advanced biomedical research. The most common protocol for cell cycle analysis is lengthy and requires ethanol for cell fixation, RNase A to remove double-stranded RNA, and propidium iodide () to label DNA. Here, we compare this routine, tedious procedure to a technique that allows quick fluorescence labeling of cellular DNA content with violet laser-excited nim-da, thereby removing the need for lengthy fixation and RNAse A incubation. INTRODUCTION Flow cytometry is an ideal technique to study various cellular components, including nucleic acids, lipids, and proteins. Of the multiple flow cytometry analyses possible, cell cycle is widely utilized as a functional cellular DNA content assay. The most common protocol for cell cycle analysis requires resuspending the cells of interest for at least 2 hours in cold ethanol. After a thorough wash, the cells are then incubated with prior to acquisition on a flow cytometer. Since is an intercalating dye that binds double-stranded nucleic acids, RNA must be removed with RNase A [2, 3]. BECKMAN COULTER WHITE PAPER 2

3 Simpler techniques exist for the study of cell cycle, most notably involving 4 6,-diamidino-2-phylindole (DA). This chemical binds to A-T-rich regions of the DNA and negates any need for RNase A treatment. DA can be cell-permeant at saturating concentrations, and there is no requirement for a lengthy alcohol fixation [4]. Nuclear Isolation Medium-DA (nim-da) contains both DA and the detergent NP-40, allowing for simultaneous cell plasma membrane removal, and DA DNA binding. Darzynkiewicz et al [1] determined that the staining equilibrium with nim-da could occur in 5 minutes. One of the main reasons why DA is an underutilized cell cycle reagent is the misconception that this chemical is best excited by a UV laser, representing a less common flow cytometer configuration as well as a more significant laboratory expense. The Beckman Coulter Gallios* flow cytometer has a 40 mw, solid state 405 nm violet laser that allows for efficient excitation of even the most dim violet fluors. The replacement of DA as the fluorescent DNA dye in cell cycle analysis for the more commonly used would allow significant time and cost savings. To test whether nim-da can replace during cell cycle analysis, we compared the cell cycle of multiple myeloma and leukemia cell lines treated with busulfan, a DNA alkylating drug used as part of preconditioning therapy for patients undergoing hematopoietic stem cell transplantation [5, 6], while examining the ability of the violet laser in Gallios to excite DA. MATERIAL AND METHODS Cells and Drugs H929, J45.01, and B5/Bu250 6 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/ml streptomycin at 37 C in a humidified atmosphere of 5% CO 2 in air. Busulfan was freshly dissolved in DMSO immediately prior to cellular drug exposure. Cell Cycle Analysis Cells in logarithmic growth phase (1x10 6 cells/ml) were incubated for 48 hours with increasing doses of busulfan at 37 C. Control cells were treated with solvent alone. Following incubation with busulfan, the cells to be prepared for cell cycle analysis with were centrifuged and resuspended in 70% ethanol overnight at 20 C. Fixed cells were centrifuged, washed with PBS, and treated with 500 U/mL RNAse A for 30 minutes at 37 C. After addition of 50 µg/ml, the cells were stabilized for 1 hour prior to analysis by flow cytometry. Cells for cell cycle analysis using nim-da were resuspended in 200 µl of nim-da. Cells were gently vortexed, kept at room temperature for 5 minutes, and immediately analyzed by flow cytometry.. BECKMAN COULTER WHITE PAPER 3

4 Flow Cytometric Analysis of Cell Cycle The cellular DNA content of at least 10,000 cells was analyzed on a Gallios flow cytometer. Doublets were discriminated based on linear amplification of area versus peak (height) or linear amplification of nim- DA area versus DA peak (height). and nim-da voltage was set to a mean channel of 300 for G1. The proportion of cells in the different phases of cell cycle was determined using the ModFit Cell Cycle Analysis software for statistical analysis or the Kaluza* Analysis software. RESULTS Cell cycle analysis performed with nim-da is comparable to Clinically used since the 1950s, busuflan acts to block DNA replication forks by introducing interstrand DNA crosslinks and ultimately disabling the repair of proliferating cells. Known to arrest cells in G2 and induce apoptosis [5, 6], this drug was selected to compare the and DA cell cycle analysis protocols. To this end, the following cell lines were treated with busulfan, or left untreated: H929, myelomic B lymphocytes; J45.01, Jurkat T lymphocytes originating from an acute leukemia; and B5/Bu250 6, a busulfan-resistant chronic myeloid leukemia cell line. After exposure to busulfan, the cells were prepared for or DA staining. As seen in Figures 1-3, the cell-cycle profiles obtained using nim-da are comparable with using in terms of the percentage of DNA content in each phase of cell cycle. Busulfan predictably acted to arrest cells in the G2 phase while increasing the percentage of cells in the subg1/apoptosis. J45.01 cells demonstrated the highest variations between and nim-da staining (observation from three independent experiments, data not shown). These data suggest that 1) nim-da staining is as equally effective as staining in cell cycle analysis, and 2) the violet laser in the Gallios flow cytometer is sufficient for excitation. Cell lines of interest must be validated first, as not all cell membranes might be as permeant to nim-da. Additionally, the ability of the violet laser on a non-gallios flow cytometer must be tested to confirm its capability to excite nim-da. BECKMAN COULTER WHITE PAPER 4

5 Figure 1: H929 Cell line nim-da nim-da nim-da Figure 2: J45.01 Cell line nim-da nim-da nim-da BECKMAN COULTER WHITE PAPER 5

6 Figure 3: B5/Bu250 6 Cell line nim- DA nim- DA nim- DA DA stability is evident even after 24 hours The cell cycle analysis protocol requires ethanol fixation prior to DNA content labeling. Therefore, these cells can be reanalyzed at great lengths of time post-staining. Nim-DA binds to unfixed cells, and, therefore, the stability is not well understood. In order to confirm the stability of DA after the initial acquisition, we reacquired the samples 24 hours after staining. To this end, H929, J45.01, and B5/Bu250 6 cells were treated with 0, 10, or 30 µg/ml busulfan and then fluorescently labeled with nim-da. Cells were acquired and the proportion of cells in the SubG1/Apoptosis, G1, S, and G2 phases were analyzed. As shown in Figure 4, B5/Bu250 6 cells demonstrated no difference within 24 hours of acquisition in the percentages of cells in the different phases of cell cycle, even without fixation. The procedure described here demonstrates an efficient and speedy protocol for analyzing cell cycle with results that are accurately comparable to the industry standard methodology. DA fluorescence was easily observed with violet laser excitation, thereby negating the need for an expensive UV laser. When adopting cell cycle analysis based on DA fluorescence, special attention needs to be made to the instrument configuration since not all violet lasers might be capable of efficient DA excitation. Unlike many common flow cytometers that utilize fiber optics for laser light delivery, the Gallios instrument efficiently delivers light directly to the flow cell losing little to no power along its path, making the Gallios an ideal instrument for violet laser functional studies. BECKMAN COULTER WHITE PAPER 6

7 Figure 4: B5/Bu250 6 Stability Initial Run 24 hours later nim-da Control nim-da 10 µg/ml Busulfan nim-da 30 µg/ml Busulfan REFERENCES 1. Darzynkiewicz, Z, Crissman, H, Jaccobberger, JW. Cytometry Part A A: Van Dilla, MA, Truiullo, TT, Mullaney, PF, Coultex, JR. Science (3872): Krishan, A. The Journal of Cell Biology : Pozarowski, P, Darzynkiewicz, Z. Methods in Molecular Biology : Ciurea, SO, Andersson, BS. Biology of Blood and Marrow Transplantation (5): Valdez, BC, Li, Y, Murray, D, Champlin, RE, Andersson, BS. Biochem. Pharmacol (2): * Gallios and Kaluza are for research use only. Not for use in diagnostic procedures BECKMAN COULTER WHITE PAPER 7

8 NOTES The results demonstrated in this application sheet represent those generated on the Beckman Coulter Gallios Flow Cytometer. As differences exist in the performance between analyzers, the authors cannot guarantee similar results with the use of other flow cytometers. REAGENT DETAILS Reagent Supplier Order Details Status nim-da BCI RUO propidium iodide Invitrogen P3566 RUO BECKMAN COULTER WHITE PAPER 8 BR-18940A B