PARAFORMALDEHYDE FIXATION OF HEMATOPOIETIC CELLS FOR QUANTITATIVE FLOW CYTOMETRY (FACS) ANALYSIS 1

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1 Journal oflmmunological Methods, 47 (1981) Elsevier/North-Holland Biomedical Press PARAFORMALDEHYDE FIXATION OF HEMATOPOIETIC CELLS FOR QUANTITATIVE FLOW CYTOMETRY (FACS) ANALYSIS 1 L.L. LANIER 2 and N.L. WARNER 3 Immunobiology Laboratories, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, U.S.A. (Received 22 May 1981, accepted 10 July 1981} The objective of this study was to find a simple method to preserve cells for subsequent flow cytometry (FACS) analysis. We determined that fixation in 1% paraformaldehyde--o.85% saline solution did not significantly alter the Coulter volume, light scatter or fluorescence properties of the cells. This method was suitable for all cell types analyzed, including mouse, human and rat lymphoid cells, erythrocytes and transformed cell lines. Furthermore, fixed cells, previously stained with fluorescein conjugated antibodies, could be stored at 4 C in the dark for at least a week prior to FACS analysis. This method should prove useful to those who work with in vivo derived specimens or have limited access to flow cytometry facilities. INTRODUCTION Recent advances in flow cytometry technology have expanded the use of these techniques into a variety of fields including immunology, cytogenetics, bacteriology, parasitology, oncology and many other areas. A major application of flow cytometry involves the analysis of cells by immunofluorescence, using FITC or TRITC conjugated antibodies. Using specific antibodies, it is possible to very precisely and objectively analyze cell surface antigens both quantitatively and qualitatively using flow cytometry measurement {Steinkamp et al., 1973; Herzenberg and Herzenberg, 1978; Warner et al., 1979). In these studies, cells are routinely stained by conventional direct or indirect immunofluorescence techniques, then the freshly stained, viable cells are analyzed using a fluorescence activated cell sorter. However, it is often impossible to schedule use of the flow cytometry facilities on the same day as the biological materials are available for staining (e.g., primary or in vivo transplanted animal tumors, patient specimens, etc.). In these situations, it 1 Supported by NIH Grants CA23770 and CA Fellow in Cancer Research supported by Grant DR6-344-FT of the Damon Runyon- Walter Winchell Cancer Fund. 3 Current address: Becton Dickinson and Company, Monoclonal Research Center, 506 Clyde Avenue, Mountain View, CA, U.S.A /81/ /$ Elsevier/North-Holland Biomedical Press

2 26 would be of considerable benefit to be able to stain the cells, and preserve them until such time as it is convenient to carry out flow cytometry analysis. In this report, we describe a simple method for fixation of cells, which does not significantly alter the Coulter volume, light scatter or fluorescence properties of cells. MATERIALS AND METHODS Cells BALB/c ByJ spleens were dissociated in cold PBS (ph 7.2, 0.1 M phosphate) and a single cell suspension prepared. Erythrocytes were lysed by hypotonic shock in distilled water and the cells washed extensively. Antisera and monoclonal antibodies FITC sheep anti-mouse ~chain serum was prepared by this laboratory and was absorbed extensively with thymocytes prior to use. Monoclonal rat antimouse Thy-l.2 (30-H12) antibody was kindly provided by Dr. L. Herzenberg and J. Ledbetter (Stanford University). Monoclonal anti-ia.7 antibody was generously provided by Dr. D. McKean {Mayo Clinic). FITC mouse (SJL) anti-rat ~ monoclonal antibody was prepared by this laboratory (Lanier et al., 1981) and FITC protein A was obtained from Pharmacia Fine Chemicals, Piscataway, NJ. Immunofluorescence staining and fixation Cells were stained by standard direct or indirect immunofluorescence techniques, as described elsewhere (Lanier and Warner, 1981). Briefly, 106 cells in 50 tzl PBS were incubated for 15 min at 4 C with a pre<letermined optimal amount of first or second stage antibody, the cells washed extensively and analyzed by flow cytometry. In experiments where fixation was employed, the cells were washed twice in large volumes of cold PBS after the final incubation with antibody. One milliliter of cold 1% (w/v) paraformaldehyde (Eastman Kodak, Rochester, NY) solution in 0.85% saline was placed into the cell pellet and the cell suspension vortexed immediately. Samples were stored in the dark at 4 C until analysis. Flow cytometry analysis The cells were analyzed using either a Becton Dickinson FACS III system (B-D, Sunnydale, CA) (Herzenberg and Herzenberg, 1978; Lanier and Warner, 1981) or the flow cytometry facilities at the Los Alamos Scientific Laboratories (Steinkamp et al., 1973; Warner et al., 1979). Analysis was performed using the 488 nm laser line, and the instruments were calibrated daily using either glutaraldehyde fixed chicken erythrocytes or fluorescent latex particles (Herzenberg and Herzenberg, 1978).

3 27 RESULTS AND DISCUSSION The objective of this study was to select a simple method to preserve cells stained with fluorescein conjugated antibodies for subsequent analysis by flow cytometry. The basic requirements were: (1) the fixation process must be simple to perform using readily available reagents; (2) fixation must not significantly alter the light scatter, Coulter volume or fluorescence properties of the cells and; (3) the process must allow for at least several days storage of the cells prior to analysis. These criteria were satisfied by a method using paraformaldehyde fixation. Using the LASL flow cytometry system which allows correlated measurements of Coulter volume, light scatter and fluorescence intensity, fresh viable spleen cells were compared with cells fixed several hours earlier in 1% paraformaldehyde saline solution. In Fig. 1, we present the Coulter volume and fluorescence histogram profiles of BALB/c splenocytes stained with anti-~ or anti-ia antibodies. Fixed cells (right panel) were essentially identical to fresh, viable cells (left panel). Thus, this experiment demonstrates that fixation in paraformaldehyde-saiine solution does not significantly alter the Coulter volume, fluorescence or light scatter (not shown) properties of splenocytes. Preservation of not only the fluorescence properties, but also light scatter and Coulter volume properties is quite significant. Low angle light scatter measurements are routinely used to discriminate live versus dead cells in a sample preparation, and as an estimate of cell size (Herzenberg and Herzenberg, 1978). Moreover, Coulter volume measurements can be used to determine the relationship between cell surface area and fluorescence intensity {i.e., surface antigen density) (Jett et al., 1980}. In these studies, we also have employed a variety of other cell types, including mouse erythrocytes, murine peripheral blood lymphocytes, murine thymocytes, murine lymphoid and monocyte/macrophage cell lines, human peripheral blood cells, human lymphoid cell lines and rat lymphoid cells. In all cases, analysis of paraformaldehyde fixed cells has proven satisfactory. Another aspect of this investigation was to determine whether fixed cells could be stored for at least several days prior to flow cytometry analysis. In these experiments, splenocytes, stained with anti-thy-l.2 monoclonal antibody were fixed in 1% paraformaldehyde-saline solution, and analyzed over a week period. As shown in Fig. 2, the fluorescence histogram profile of fresh, viable splenocytes was essential identical to fixed cells analyzed 8 days later using a B-D FACS II! system. Similar results were found using other cell types and analysis of other surface antigens {data not shown). Although we have not as yet determined the maximum storage time of fixed cells, paraformaldehyde fixed mouse splenocytes stained with fluorescein-conjugated antibodies can be stored at 4 C for at least 2 months without significant alterations in light scatter or fluorescence properties (data not shown). Furthermore, a formal study comparing concentrations of fixative or different fixatives has not been undertaken. However, we have also successfully

4 28 [ COULT[R VOLUME a FIXED C D AUTOFLUORESCENCE CONTROL 'Z. ; Fig. 1. Flow cytometry analysis of viable vs. paraformaldehyde fixed splenocytes. BALB/c splenocytes were stained using FITC-sheep anti-mouse kappa chain serum, or monoclonal anti-ia.7 antibody, followed by FITC-protein A. Cells were analyzed using the Los Alamos Scientific Laboratories flow systems either immediately after staining (.viable) or' after paraformaldehyde fixation (fixed). A and B show the Coulter volume histograms of the fresh, viable and fixed cells, respectively (x axis = Coulter channels , linear scale; y axis = number of cells). In C and D, the fluorescence histograms of unstained, control cells are presented; whereas in E--H, the fluorescence histogram of cells stained with anti-k (E, F) and anti-ia.7 (G, H) are shown (x axis = fluorescence channels , 3 decade log scale; y axis = number of cells). Note that the Coulter and fluorescence histograms of the viable and fixed splenocytes are essentially identical. Quantitative analysis of the data indicates that 42.6% of the viable cells stained positively with anti-k serum and 21% stained with anti-ia.7 monoclonal antibody. Similarly, 43.1% of the fixed cells stained with the anti4~ reagent, and 24.2% stained with anti-ia.7 monoclonal antibody. (Percentage positive cells was determined by calculating the number of cells in fluorescence channels , dividing by the total number of cells analyzed (104) and multiplying by 100.) A more detailed description of the data analysis has been presented elsewhere (Steinkamp et al., 1973; Warner et al., 1979). In these experiments the voltage and gain settings on the amplifiers were identical for all samples. used 1% formaldehyde-saline solution. Glutaraldehyde fixation was found unacceptable, in that it resulted in high immunofluorescence backgrounds. It should be noted that in all the experiments presented, the cells were fixed after immunofluorescence staining. Another obvious consideration was whether it was possible to fix unstained cells for later immunofluorescence

5 29 A B C Fig. 2. FACS analysis of fixed, stained splenocytes after storage. BALB/c splenocytes were stained with medium (control) or monoclonal anti-thy-l.2 antibody, followed by FITC mouse (SJL) anti-rat g monoclonal antibody. Cells were analyzed either immediately after staining (fresh, viable cells; A), immediately after fixation (fixed cells, day 0; B), or after storage at 4vC in the dark for 1 week (C). Analysis was performed using a FACS III system. The fluorescence histograms are displayed above (x axis = fluorescence intensity, 128 channels, linear scale; y axis = number of cells). Voltage and gain settings on the amplifier were identical for all samples analyzed (900 V, 16 1 gain). In these figures, the histogram of the stained cells was superimposed over the histogram of the control (left peak). Note that there is a very slight drop in fluorescence intensity after 1 week storage. However, quantitative analysis revealed that a similar percentage of cells were scored Thy-l.2!n all 3 situations (fresh (A) = 59%; fixed, day 0 (B) = 58%; fixed 7 days = 54%). Percentage positive cells was determined by calculating the number of cells in fluorescence channels , dividing by the total number of cells analyzed (104), and multiplying by 100. A more detailed description of the FACS analysis has been presented elsewhere (Lanier and Warner, 1981). staining. All attempts to fix cells prior to staining were unsuccessful and resulted in high autofluorescence backgrounds. In summary, we have described a very simple fixation method, using 1% paraformaldehyde-saline solution, which allows storage of cells for later analysis by flow cytometry. It should be of considerable value to those who frequently work with in vivo derived specimens or have limited access to flow cytometry facilities. Fixation of cells also may be desirable to prevent capping or shedding of surface proteins in the interim between staining and cell analysis. ACKNOWLEDGEMENTS We thank Mrs. Eva Barry for her superb management of the cell culture facilities, Mr. Jerome Zawadzki for expert assistance with the FACS analysis, Mr. John Martin of the Los Alamos Scientific Laboratories for helpful assistance, and Miss Sharon Novaco for excellent secretarial assistance.

6 30 REFERENCES Herzenberg, L.A. and L.A. Herzenberg, 1978, in: Handbook of Experimental Immunology, 3rd edn, ed. D.M. Weir (Blackwell Scientific, London) p Jett, J.H., A.P. Stevenson, N.L. Warner and J.F. Leafy, 1980, Flow Cytometry 4,215. Lanier, L.L. and N.L. Warner, 1981, J. Immunol. 125,626. Lanier, L.L., G.A. Gutman, D.E. Lewis, S. Griswold and N.L. Warner, 1981, Hybridoma, in press. Steinkamp, J.A., M.L. Fulwyler, J.R. Coulter, R.D. Hiebert, J,L. Horney and P.L. Mulhaney, 1973, Rev. Sci. Instrum. 44, Warner, N.L., M.J. Daley, J. Richey and C. Spellman, 1979, Immunol. Rev. 48,197.