Journal of Clinical Laboratory Analysis 4:324-327 (1990)
Simultaneous Measurement of Neutrophil, Lymphocyte, and Monocyte Glutathione by Flow Cytometry Robert B. Sc0tt,'9*9~James M. C o l l i n ~Sina , ~ Matin,' Frances White: and Paul S. Swerdlow' Departments of 'Medicine, 2Pathology;and 3Biochemistryand Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia A flow cytometric method for quantitation delineating those cells by their light-scatter of glutathione (GSH) was applied to simulta- characteristics,utilizing dual-angle light scatneous analysis of the major leukocyte types ter for discrimination. By this means, GSH in peripheral blood. Cellular thiols (predomi- contents of 12.5 k 2.0 nmol/107neutrophils, nately GSH) were stained with monochloro- 14.5 ? 2.7 nmol/107monocytes, and 5.0 f bimane (MCIB), and thiol fluorescence was 1.O nmol/107 lymphocytes were found. The measured with a flow cytometer. The fluores- results obtained for neutrophils with the flow cence of the thiols closely reflected the GSH cytometer were virtually identical with those content, as measured by a specific glutathi- obtained with chemical assay in purified samone reductase assay. Fluorescence of indi- ples of neutrophils, indicating the validity of vidual cell types could be measured after the flow cytometric method. Key words:
Leukocytes, rnonochlorobirnane,peripheral blood, fluorescence, light scatter
cross cell membranes and are not fluorescent until they react with thiol groups (14). Leukocyte subtypes of peripheral Glutathione(GSH),the tripeptide gamrna-glutamyl-cysteinyl- blood were identified in flow cytometry by their unique lightglycine, is an important cytosolic thiol component of many scatter properties, and the GSH fluorescence was quantitated. cells. GSH serves as a protection against oxidant stress through glutathione peroxidase and also serves as substrate for GSH MATERIALS AND METHODS S-transferase, which catalyzes the conjugation with GSH and excretion from the cell of electrophilic xenobiotics and po- Preparation of Leukocyte Suspensions tentially toxic chemicals. Blood collected in EDTA was mixed with equal volumes Among blood cells, GSH has been most studied in erythof 3% dextran in phosphate-buffered saline (PBS), pH 7.4; rocytes (1-4), and it has been investigated to a lesser extent after 50 min of sedimentation at unit gravity, mixed white in leukocytes of mice (5) and humans (6,7). The lack of studcells were collected by centrifuging the plasma at 200 g for ies of leukocytes can in part be ascribed to the relative diffi15 min. The pellet was subjected to two brief (less than 30 culty involved in the effective separation of the different sec) exposures to distilled water to lyse remaining erythroleukocyte types, the relative paucity of monocytes among the cytes, and tonicity was restored each time with one-tenth volmore prevalent neutrophils and lymphocytes, and the gen- ume of 2.5 M sucrose prior to a final wash with PBS. Donors eral lack of understanding of the physiologic role of GSH in were healthy ambulatory individuals, usually laboratory staff these cells. or medical students. Cells were found to be over 90% viable In order to facilitate study of GSH in blood cells, we in- by Trypan blue dye exclusion. Neutrophils were also purified vestigated the possibility of estimating GSH in mixtures of from blood of young healthy donors by centrifuging the cells leukocytes simultaneously by flow cytometry. Others (8- 12) from erythrocyte-depleted plasma through Ficoll-Hypaque , have shown that fluorescent label of thiols can be used suc- density 1.077, and collecting the neutrophils from the pelcessfully in flow cytometry of cultured cells. With such a lets. They were washed in PBS to remove Ficoll-Hypaque technique, it would then be possible to measure GSH in the prior to assay of GSH. major leukocyte subsets simultaneously and to perform the assay on small blood samples. We used monochlorobimane (MClB) as a fluorescent label of the GSH. MClB conjugates with protein sulfhydryls to the extent of 2%, which is less Received January 8, 1990; accepted April 9, 1990. than monobromobimane which produces 10% conjugation Address reprint requests to Robert B . Scott, M.D., Medical College of Virwith protein sulfiydryls (13). The halogenated bimanes readily ginia, Richmond, VA 23298-0214.
INTRODUCTION
0 1990 Wiley-Liss, Inc.
GSH in Peripheral Blood Leukocytes
Chemical Determinationof GSH Cell suspensionswere extracted with equal volumes of 1 M perchloric acid (PCA) containing 10 mM EDTA. GSH was determined on the extract by the glutathione reductase cycling assay of Tietze, which measures both reduced and oxidized GSH as “total” GSH (15).
Preparation of Cultured Cells HL60 cells were grown in RPMI 1640 medium with 10% defined supplemented bovine calf serum (Hyclone), added glutamine, nonessential amino acids, and sodium pyruvate. HeLa cells were maintained in Joklik’s modified minimal essential medium containing 10% fetal calf serum and 1.25% Fungizone and routinely monitored for mycoplasma as previously described (16).
Staining of the Cells MClB was obtained from Molecular Probes (Eugene, OR) and kept frozen as a 10 mM solution in acetonitrile and protected from light. For staining, aliquots were diluted in PBS. Cell suspensions containing 4 x lo6cells were added to 2 ml of 10 pM MClB in buffer and incubated in the dark at 37°C. for 20 min.
Flow Cytometry
325
GSH was paralleled by a concomitant decrease in MClB fluorescence. The results of one of two similar experiments are shown in Figure 1. At DEM concentrations up to 5 mM, the GSH content of the cells (shown on the horizontal axis) was linearly related to the mean channel number of the fluorescence intensity recorded in the flow cytometer (r = 0.9418).
Light-Scatter Characteristics of Peripheral Blood Leukocytes In previous work, we demonstrated that leukocytes show different characteristicsof forward-angle light scatter, which permit their separation (17). The present method utilizes the simultaneous measurement of both forward and 90” light scatter. Light-scatter profiles of the three populations were defined and electronic gates were constructed around each population for further analysis. Cells from each of these areas were recovered by electronic cell sorting and concentrated on microscope slides for staining and microscopy. The areas delineated were shown to be lymphocytes, monocytes, and neutrophils. The cell populations were nearly completely separated, such that the purity of the neutrophils was typically 95% or greater, the monocytes 85% or greater, and the lymphocytes 95%-99%. The other concentrations of cells in the two-dimensionallight-scatter plot were mixtures of cells and may represent less specific changes in light-scatter properties associated with devitalization of cells.
The stained cells were excited at 360 nm and both forward Calibration of the GSH Cellular Measurements and 90” light scatter were measured. Fluorescence was siFluorescence intensity, which reflects the GSH content of multaneouslymeasured, using a 420-nm-longpass filter. Corthe cells, is determined from the mean intensity over the disrections for autofluorescence were made from measurements tribution of the cells in question. A cell suitable for calibrawith unstained cells. tion of the fluorescence:GSH ratio would be of a readily Based on electronic selection of discrete populations acavailable pure population of cells in suspension, with a unicording to their dual-angle light-scatter characteristics, cells were sorted for morphologic analysis and stained with Wright’s blood stain. L
RESULTS Relationship of MClB Staining to Cellular GSH In a preliminary study, it was shown that blocking cellular thiols with N-ethylmaleimide (200 pM) prior to staining the cells with MClB inhibited 98% of the fluorescence. Thus, almost all of the observed fluorescence with MClB was thiolrelated. GSH is believed to account for the vast majority of thiols in cells, with minor proportions found in protein thiols and in metabolites such as coenzyme A. Correction was also made for autofluorescenceof cells by subtractingfluorescence measured in unstained cell suspensions. In order to determine the relationship of MClB fluorescenceand GSH content, Hela cells were incubated in various concentrations of diethylmaleate (DEM), which forms an adduct with GSH and effectively “depletes” cellular GSH. In this way, it could be determined whether the decrease in specifically measured
GSH (nMol/Million Cells) Fig. 1. Linear relationship of fluorescence of monochlorobimane-stained HeLa cells to chemically quantitated glutathione in the same cells (r = 0.9418). GSH content was varied by incubation of the cells in various concentrations of diethylmaleate (DEM) up to 5 mM, which depleted cellular GSH.
326
Scottetal.
form size distribution and a concentration of GSH at least as high as the leukocyte with the largest GSH content, i.e., monocytes. It would also have consistent GSH concentration in repeat samples. Several types were tested, including Hela cells, K562 leukemia cells, L1210 cells, and HL60 cells. Of these, HL60 cells were found to fit these criteria best and were chosen for subsequent analyses. GSH content of HL60 cells, measured by a specific glutathione reductase method in nine separate culture passages, was 21.9 & 2.1 nmoV107cells. The HL60 samples were from exponentiallygrowing culturesat concentrationof0.3-0.9 X lo6 cells/ml. When HL60 cells were stained with MClB and studied by flow cytometry, the mean channel number, representingthe fluorescenceintensity, was 42.8 & 5.3 in five separate samples. From the relationship between GSH content and MClB fluorescence, the GSH content of the leukocytes could be calculated after determining the mean channel number (intensity) of the MClB fluorescence for each cell type. As shown in Table 1, the mean channel number for the neutrophils was 24.5 & 3.9; for monocytes it was 28.4 2 5.3 (SD), and for lymphocytes it was 9.7 & 2.0. Comparing these fluorescence intensities with that of the HL60 cells, the GSH content of the leukocytes was calculated to be 12.5 & 2.0 nmoV lo7neutrophils, 14.5 ? 2.7 nmol/107monocytes, and 5.0 & 1.O nmoV107 lymphocytes. The accuracy of the flow cytometer measurements was also assessed by comparing the determinations above with normal values for GSH content in samples of normal neutrophils purified by dextran sedimentation of the erythrocytes and isolation of the neutrophils in density gradients of FicollHypaque. The results (Table 1) show that the isolated neutrophils had a GSH content of 12.3 f 2.5 nmol/107 cells (n = 12), which is virtually identical to the results from the flow cytometry determination.
DISCUSSION Separationof individual types of blood leukocytes for study is laborious and somewhat difficult. Neutrophil leukocytes, the most prominent type in the circulating (peripheral) blood, can be isolated by centrifugationthrough dense solutions such as Ficoll-Hypaque, which was used in these studies. The frequently used Ficoll-Hypaque separation of mononuclearcells
separates a population that is a mixture of lymphocytes and monocytes, which share a similar buoyant density. These then must be further separated by adherence of the monocytes to glass or plastic surfaces or removal of lymphocyte populations with cell-specific antibodies. Any quantitative assay that can obviate these tedious procedures is of great value. The procedure reported here illustrates a relatively simple means of quantitating GSH in crude mixtures of leukocytes, taking advantage of the technology now available with flow cytometry. The two-dimensional light-scatter patterns readily delineate populations of virtually pure neutrophils, lymphocytes, and monocytes. The electronic cell-sorting capability of the cytometer also permits the morphologic determination of the locations of the different cells types. In addition to the patterns representing the three major cell types, other light-scatter images were present in the two-dimensional arrays. These represented various mixtures of cells of altered light-scatter characteristics. Their significance was not investigated. The results reported herein show that (a) the fluorescence probe labels virtually all of the cell thiols (of which the vast majority represent GSH); (b) fluorescence intensity closely reflects the measured content of GSH as the cells are depleted of GSH; and (c) the results in the fluorescence assay are identical to the results of specific chemical determinations of GSH in purified suspensions of neutrophils. The GSH content calculated for lymphocytesand monocytes is proportional to their relative cell sizes. Also, the ratio of neutrophil GSH to lymphocytes GSH (2.5:l) is comparable with the 2.8:l ratio found by Carmichael et al. (5) for mouse granulocytes and lymphocytes. Having determined the reliability of this flow cytometric assay for samples of human peripheral blood, we believe the assay should be adaptable to small samples of human or animal blood as well as mixed cell populations recovered from tissues.
ACKNOWLEDGMENTS We wish to thank Annie Chu, Page Wirt, and Patricia Nelms for expert technical assistance. Supported by Grant 5 KO7 AGO0404 from the National Institute of Aging (R.B.S.).
TABLE 1. Comparison of GSH Content of Leukocytes Measured Chemically in Purified Preparations of Cells and Measured in Flow Cytometry by Fluorescence of MCIB-GSH Measured GSH* ( n m 0 ~ 1 0cells) ~ Mean SD Number
Fluorescence (mean channel no.)
Calculated GSHt (nmov1o7 cells)
PMN
HL60
HL60
PMN
MONO
LYMPH
12.3
21.9 k2.1 9
42.8 25.3
24.5 k3.9
5
10
28.4 k5.3 10
? 2.5
12
PMN
MONO
LYMPH
* 2.0
12.5 22.0
2 1.0
10
10
14.5 k2.1 10
9.7
PMN, polymorphonuclear neutruphil; MONO, monocyte; LYMPH, lymphocyte. *GSH measured biochemically in purified neutrophil preparations and cultures of HL60 cells. tGSH calculated from MClB fluorescence of cells compared with fluorescence of HL60 cells with known GSH content
5.0 10
GSH in Peripheral Blood Leukocytes REFERENCES 1. Agar NS, Lewis GBH: Effect of halothane and enflurane anesthesia on the level of reduced glutathione in human red blood cells. Anesrh Intens Cure 8:356-358, 1980. 2. Board PG:Transport of glutathione S-conjugate from human erythrocytes. FEBSLert 124163-165, 1981. 3. Dixit R, Das M, Seth PK, Mukhtar H: Interaction of acrylamide with glutathione in rat erythrocytes. ToxicolLen 23:291-298, 1984. 4. Imanishi H, Nakai T, Abe T, Takino T Glutathione metabolism in red cell aging. Mech Age Devel32:57-62, 1985. 5 . Carmichael J, Adams DJ, Ansell J, Wolf CR: Glutathione and glutathione transferase levels in mouse granulocytes following cyclophosphamide administration. Cancer Res 46:735-739,1986. 6. Burchill BR, Oliver JM, Pearson CB, Leinbach ED, Berlin RD: Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclearleukocytes.J Cell Biol76:439-447, 1978. 7. Rajkovic IA,Williams R: Inhibition of neutrophil function by hydrogen peroxide. Biochem Phurmacol34:2083-2090, 1985. 8. Poot M, Verkerk A, Koster JF, Jongkind J F De novo synthesis of glutathione in human fibroblasts during in vitro ageing and in some metabolic diseases as measured by a flow cytometric method. Biochim BiophysActu 883580-584, 1986. 9. Treumer J, Valet G: Flow-cytometric determinationof glutathione alterations in vital cells by o-phthaldialdehyde(OPT)staining. Exp Cell Res 163518-524,1986.
327
10. Cook JA, Pass HI, Russo A, Iype S, Mitchell JB: Use of monochlorobimane for glutathionemeasurements in hamster and human tumor cell lines. IntJRudOncolBiol Phys 16:1321-1324, 1989. 11. Shrieve DC, Bump EA, Rice GC: Heterogeneityof cellular glutathione among cells derived from a murine fibrosarcoma or a human renal cell carcinoma detected by flow cytometric analysis. J Biol Chem 263: 14107-141 14,1988. 12. Lee FYF, Siemann DW: Isolation by flow cytometry of a human ovarian tumor cell subpopulationexhibiting a high glutathione content phenotype and increased resistance to adriamycin. Int J Rud Oncof Biot PhyS 16:1315-1319, 1989. 13. Rice GS, Bump EA, Shrieve DC, Lee W, Kovacs M: Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: Some applicationsto radiation and drug resistance in vitro and in vivo. CancerRes 46:6105-6110, 1986. 14. Kosower NS, Kosower EM, Newton GL, Ranney HM: Bimane fluorescent labels: Labeling of normal human red cells under physiologicconditions. Proc Nut Acud Sci USA 76:3382-3386, 1979. 15. Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem 27: 502-522, 1969. 16. Collins JM: Rates of DNA synthesis during S-phase of HeLa cells. J Biol Chem 2533570-8577,1978. 17. Scott RB, Grogan WM, Collins JM: Separation of rabbit marrow precursor cells by combined isopycnic sedimentation and electroniccellsorting. Blood51:1137-1148, 1978.
Simultaneous Measurement of Neutrophil, Lymphocyte, and Monocyte Glutathione by Flow Cytometry Robert B. Sc0tt,'9*9~James M. C o l l i n ~Sina , ~ Matin,' Frances White: and Paul S. Swerdlow' Departments of 'Medicine, 2Pathology;and 3Biochemistryand Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia A flow cytometric method for quantitation delineating those cells by their light-scatter of glutathione (GSH) was applied to simulta- characteristics,utilizing dual-angle light scatneous analysis of the major leukocyte types ter for discrimination. By this means, GSH in peripheral blood. Cellular thiols (predomi- contents of 12.5 k 2.0 nmol/107neutrophils, nately GSH) were stained with monochloro- 14.5 ? 2.7 nmol/107monocytes, and 5.0 f bimane (MCIB), and thiol fluorescence was 1.O nmol/107 lymphocytes were found. The measured with a flow cytometer. The fluores- results obtained for neutrophils with the flow cence of the thiols closely reflected the GSH cytometer were virtually identical with those content, as measured by a specific glutathi- obtained with chemical assay in purified samone reductase assay. Fluorescence of indi- ples of neutrophils, indicating the validity of vidual cell types could be measured after the flow cytometric method. Key words:
Leukocytes, rnonochlorobirnane,peripheral blood, fluorescence, light scatter
cross cell membranes and are not fluorescent until they react with thiol groups (14). Leukocyte subtypes of peripheral Glutathione(GSH),the tripeptide gamrna-glutamyl-cysteinyl- blood were identified in flow cytometry by their unique lightglycine, is an important cytosolic thiol component of many scatter properties, and the GSH fluorescence was quantitated. cells. GSH serves as a protection against oxidant stress through glutathione peroxidase and also serves as substrate for GSH MATERIALS AND METHODS S-transferase, which catalyzes the conjugation with GSH and excretion from the cell of electrophilic xenobiotics and po- Preparation of Leukocyte Suspensions tentially toxic chemicals. Blood collected in EDTA was mixed with equal volumes Among blood cells, GSH has been most studied in erythof 3% dextran in phosphate-buffered saline (PBS), pH 7.4; rocytes (1-4), and it has been investigated to a lesser extent after 50 min of sedimentation at unit gravity, mixed white in leukocytes of mice (5) and humans (6,7). The lack of studcells were collected by centrifuging the plasma at 200 g for ies of leukocytes can in part be ascribed to the relative diffi15 min. The pellet was subjected to two brief (less than 30 culty involved in the effective separation of the different sec) exposures to distilled water to lyse remaining erythroleukocyte types, the relative paucity of monocytes among the cytes, and tonicity was restored each time with one-tenth volmore prevalent neutrophils and lymphocytes, and the gen- ume of 2.5 M sucrose prior to a final wash with PBS. Donors eral lack of understanding of the physiologic role of GSH in were healthy ambulatory individuals, usually laboratory staff these cells. or medical students. Cells were found to be over 90% viable In order to facilitate study of GSH in blood cells, we in- by Trypan blue dye exclusion. Neutrophils were also purified vestigated the possibility of estimating GSH in mixtures of from blood of young healthy donors by centrifuging the cells leukocytes simultaneously by flow cytometry. Others (8- 12) from erythrocyte-depleted plasma through Ficoll-Hypaque , have shown that fluorescent label of thiols can be used suc- density 1.077, and collecting the neutrophils from the pelcessfully in flow cytometry of cultured cells. With such a lets. They were washed in PBS to remove Ficoll-Hypaque technique, it would then be possible to measure GSH in the prior to assay of GSH. major leukocyte subsets simultaneously and to perform the assay on small blood samples. We used monochlorobimane (MClB) as a fluorescent label of the GSH. MClB conjugates with protein sulfhydryls to the extent of 2%, which is less Received January 8, 1990; accepted April 9, 1990. than monobromobimane which produces 10% conjugation Address reprint requests to Robert B . Scott, M.D., Medical College of Virwith protein sulfiydryls (13). The halogenated bimanes readily ginia, Richmond, VA 23298-0214.
INTRODUCTION
0 1990 Wiley-Liss, Inc.
GSH in Peripheral Blood Leukocytes
Chemical Determinationof GSH Cell suspensionswere extracted with equal volumes of 1 M perchloric acid (PCA) containing 10 mM EDTA. GSH was determined on the extract by the glutathione reductase cycling assay of Tietze, which measures both reduced and oxidized GSH as “total” GSH (15).
Preparation of Cultured Cells HL60 cells were grown in RPMI 1640 medium with 10% defined supplemented bovine calf serum (Hyclone), added glutamine, nonessential amino acids, and sodium pyruvate. HeLa cells were maintained in Joklik’s modified minimal essential medium containing 10% fetal calf serum and 1.25% Fungizone and routinely monitored for mycoplasma as previously described (16).
Staining of the Cells MClB was obtained from Molecular Probes (Eugene, OR) and kept frozen as a 10 mM solution in acetonitrile and protected from light. For staining, aliquots were diluted in PBS. Cell suspensions containing 4 x lo6cells were added to 2 ml of 10 pM MClB in buffer and incubated in the dark at 37°C. for 20 min.
Flow Cytometry
325
GSH was paralleled by a concomitant decrease in MClB fluorescence. The results of one of two similar experiments are shown in Figure 1. At DEM concentrations up to 5 mM, the GSH content of the cells (shown on the horizontal axis) was linearly related to the mean channel number of the fluorescence intensity recorded in the flow cytometer (r = 0.9418).
Light-Scatter Characteristics of Peripheral Blood Leukocytes In previous work, we demonstrated that leukocytes show different characteristicsof forward-angle light scatter, which permit their separation (17). The present method utilizes the simultaneous measurement of both forward and 90” light scatter. Light-scatter profiles of the three populations were defined and electronic gates were constructed around each population for further analysis. Cells from each of these areas were recovered by electronic cell sorting and concentrated on microscope slides for staining and microscopy. The areas delineated were shown to be lymphocytes, monocytes, and neutrophils. The cell populations were nearly completely separated, such that the purity of the neutrophils was typically 95% or greater, the monocytes 85% or greater, and the lymphocytes 95%-99%. The other concentrations of cells in the two-dimensionallight-scatter plot were mixtures of cells and may represent less specific changes in light-scatter properties associated with devitalization of cells.
The stained cells were excited at 360 nm and both forward Calibration of the GSH Cellular Measurements and 90” light scatter were measured. Fluorescence was siFluorescence intensity, which reflects the GSH content of multaneouslymeasured, using a 420-nm-longpass filter. Corthe cells, is determined from the mean intensity over the disrections for autofluorescence were made from measurements tribution of the cells in question. A cell suitable for calibrawith unstained cells. tion of the fluorescence:GSH ratio would be of a readily Based on electronic selection of discrete populations acavailable pure population of cells in suspension, with a unicording to their dual-angle light-scatter characteristics, cells were sorted for morphologic analysis and stained with Wright’s blood stain. L
RESULTS Relationship of MClB Staining to Cellular GSH In a preliminary study, it was shown that blocking cellular thiols with N-ethylmaleimide (200 pM) prior to staining the cells with MClB inhibited 98% of the fluorescence. Thus, almost all of the observed fluorescence with MClB was thiolrelated. GSH is believed to account for the vast majority of thiols in cells, with minor proportions found in protein thiols and in metabolites such as coenzyme A. Correction was also made for autofluorescenceof cells by subtractingfluorescence measured in unstained cell suspensions. In order to determine the relationship of MClB fluorescenceand GSH content, Hela cells were incubated in various concentrations of diethylmaleate (DEM), which forms an adduct with GSH and effectively “depletes” cellular GSH. In this way, it could be determined whether the decrease in specifically measured
GSH (nMol/Million Cells) Fig. 1. Linear relationship of fluorescence of monochlorobimane-stained HeLa cells to chemically quantitated glutathione in the same cells (r = 0.9418). GSH content was varied by incubation of the cells in various concentrations of diethylmaleate (DEM) up to 5 mM, which depleted cellular GSH.
326
Scottetal.
form size distribution and a concentration of GSH at least as high as the leukocyte with the largest GSH content, i.e., monocytes. It would also have consistent GSH concentration in repeat samples. Several types were tested, including Hela cells, K562 leukemia cells, L1210 cells, and HL60 cells. Of these, HL60 cells were found to fit these criteria best and were chosen for subsequent analyses. GSH content of HL60 cells, measured by a specific glutathione reductase method in nine separate culture passages, was 21.9 & 2.1 nmoV107cells. The HL60 samples were from exponentiallygrowing culturesat concentrationof0.3-0.9 X lo6 cells/ml. When HL60 cells were stained with MClB and studied by flow cytometry, the mean channel number, representingthe fluorescenceintensity, was 42.8 & 5.3 in five separate samples. From the relationship between GSH content and MClB fluorescence, the GSH content of the leukocytes could be calculated after determining the mean channel number (intensity) of the MClB fluorescence for each cell type. As shown in Table 1, the mean channel number for the neutrophils was 24.5 & 3.9; for monocytes it was 28.4 2 5.3 (SD), and for lymphocytes it was 9.7 & 2.0. Comparing these fluorescence intensities with that of the HL60 cells, the GSH content of the leukocytes was calculated to be 12.5 & 2.0 nmoV lo7neutrophils, 14.5 ? 2.7 nmol/107monocytes, and 5.0 & 1.O nmoV107 lymphocytes. The accuracy of the flow cytometer measurements was also assessed by comparing the determinations above with normal values for GSH content in samples of normal neutrophils purified by dextran sedimentation of the erythrocytes and isolation of the neutrophils in density gradients of FicollHypaque. The results (Table 1) show that the isolated neutrophils had a GSH content of 12.3 f 2.5 nmol/107 cells (n = 12), which is virtually identical to the results from the flow cytometry determination.
DISCUSSION Separationof individual types of blood leukocytes for study is laborious and somewhat difficult. Neutrophil leukocytes, the most prominent type in the circulating (peripheral) blood, can be isolated by centrifugationthrough dense solutions such as Ficoll-Hypaque, which was used in these studies. The frequently used Ficoll-Hypaque separation of mononuclearcells
separates a population that is a mixture of lymphocytes and monocytes, which share a similar buoyant density. These then must be further separated by adherence of the monocytes to glass or plastic surfaces or removal of lymphocyte populations with cell-specific antibodies. Any quantitative assay that can obviate these tedious procedures is of great value. The procedure reported here illustrates a relatively simple means of quantitating GSH in crude mixtures of leukocytes, taking advantage of the technology now available with flow cytometry. The two-dimensional light-scatter patterns readily delineate populations of virtually pure neutrophils, lymphocytes, and monocytes. The electronic cell-sorting capability of the cytometer also permits the morphologic determination of the locations of the different cells types. In addition to the patterns representing the three major cell types, other light-scatter images were present in the two-dimensional arrays. These represented various mixtures of cells of altered light-scatter characteristics. Their significance was not investigated. The results reported herein show that (a) the fluorescence probe labels virtually all of the cell thiols (of which the vast majority represent GSH); (b) fluorescence intensity closely reflects the measured content of GSH as the cells are depleted of GSH; and (c) the results in the fluorescence assay are identical to the results of specific chemical determinations of GSH in purified suspensions of neutrophils. The GSH content calculated for lymphocytesand monocytes is proportional to their relative cell sizes. Also, the ratio of neutrophil GSH to lymphocytes GSH (2.5:l) is comparable with the 2.8:l ratio found by Carmichael et al. (5) for mouse granulocytes and lymphocytes. Having determined the reliability of this flow cytometric assay for samples of human peripheral blood, we believe the assay should be adaptable to small samples of human or animal blood as well as mixed cell populations recovered from tissues.
ACKNOWLEDGMENTS We wish to thank Annie Chu, Page Wirt, and Patricia Nelms for expert technical assistance. Supported by Grant 5 KO7 AGO0404 from the National Institute of Aging (R.B.S.).
TABLE 1. Comparison of GSH Content of Leukocytes Measured Chemically in Purified Preparations of Cells and Measured in Flow Cytometry by Fluorescence of MCIB-GSH Measured GSH* ( n m 0 ~ 1 0cells) ~ Mean SD Number
Fluorescence (mean channel no.)
Calculated GSHt (nmov1o7 cells)
PMN
HL60
HL60
PMN
MONO
LYMPH
12.3
21.9 k2.1 9
42.8 25.3
24.5 k3.9
5
10
28.4 k5.3 10
? 2.5
12
PMN
MONO
LYMPH
* 2.0
12.5 22.0
2 1.0
10
10
14.5 k2.1 10
9.7
PMN, polymorphonuclear neutruphil; MONO, monocyte; LYMPH, lymphocyte. *GSH measured biochemically in purified neutrophil preparations and cultures of HL60 cells. tGSH calculated from MClB fluorescence of cells compared with fluorescence of HL60 cells with known GSH content
5.0 10
GSH in Peripheral Blood Leukocytes REFERENCES 1. Agar NS, Lewis GBH: Effect of halothane and enflurane anesthesia on the level of reduced glutathione in human red blood cells. Anesrh Intens Cure 8:356-358, 1980. 2. Board PG:Transport of glutathione S-conjugate from human erythrocytes. FEBSLert 124163-165, 1981. 3. Dixit R, Das M, Seth PK, Mukhtar H: Interaction of acrylamide with glutathione in rat erythrocytes. ToxicolLen 23:291-298, 1984. 4. Imanishi H, Nakai T, Abe T, Takino T Glutathione metabolism in red cell aging. Mech Age Devel32:57-62, 1985. 5 . Carmichael J, Adams DJ, Ansell J, Wolf CR: Glutathione and glutathione transferase levels in mouse granulocytes following cyclophosphamide administration. Cancer Res 46:735-739,1986. 6. Burchill BR, Oliver JM, Pearson CB, Leinbach ED, Berlin RD: Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclearleukocytes.J Cell Biol76:439-447, 1978. 7. Rajkovic IA,Williams R: Inhibition of neutrophil function by hydrogen peroxide. Biochem Phurmacol34:2083-2090, 1985. 8. Poot M, Verkerk A, Koster JF, Jongkind J F De novo synthesis of glutathione in human fibroblasts during in vitro ageing and in some metabolic diseases as measured by a flow cytometric method. Biochim BiophysActu 883580-584, 1986. 9. Treumer J, Valet G: Flow-cytometric determinationof glutathione alterations in vital cells by o-phthaldialdehyde(OPT)staining. Exp Cell Res 163518-524,1986.
327
10. Cook JA, Pass HI, Russo A, Iype S, Mitchell JB: Use of monochlorobimane for glutathionemeasurements in hamster and human tumor cell lines. IntJRudOncolBiol Phys 16:1321-1324, 1989. 11. Shrieve DC, Bump EA, Rice GC: Heterogeneityof cellular glutathione among cells derived from a murine fibrosarcoma or a human renal cell carcinoma detected by flow cytometric analysis. J Biol Chem 263: 14107-141 14,1988. 12. Lee FYF, Siemann DW: Isolation by flow cytometry of a human ovarian tumor cell subpopulationexhibiting a high glutathione content phenotype and increased resistance to adriamycin. Int J Rud Oncof Biot PhyS 16:1315-1319, 1989. 13. Rice GS, Bump EA, Shrieve DC, Lee W, Kovacs M: Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: Some applicationsto radiation and drug resistance in vitro and in vivo. CancerRes 46:6105-6110, 1986. 14. Kosower NS, Kosower EM, Newton GL, Ranney HM: Bimane fluorescent labels: Labeling of normal human red cells under physiologicconditions. Proc Nut Acud Sci USA 76:3382-3386, 1979. 15. Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem 27: 502-522, 1969. 16. Collins JM: Rates of DNA synthesis during S-phase of HeLa cells. J Biol Chem 2533570-8577,1978. 17. Scott RB, Grogan WM, Collins JM: Separation of rabbit marrow precursor cells by combined isopycnic sedimentation and electroniccellsorting. Blood51:1137-1148, 1978.
