INFECTION AND IMMUNITY, Apr. 1985, p. 114-118 Vol. 48, No. 10019-9567/85/040114-05$02.00/0Copyright C) 1985, American Society for Microbiology

Susceptibility to Staphylococcal Alpha-Toxin of FriendVirus-Infected Murine Erythroblasts During Differentiation

SIDNEY HARSHMAN* AND MAURICE BONDURANTDepartments of Microbiology and Hematology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 31 October 1984/Accepted 31 December 1984

Splenic erythroblasts obtained from BALB/c mice infected with the anemia strain of Friend virus werecompared with "matured" cells and adult erythrocytes for their sensitivity to staphylococcal ot-toxin. Maturedcells were obtained by treating erythroblasts in culture with erythropoietin for 48 h. Sensitivity tostaphylococcal oa-toxin, measured both by release of 86Rb and by cell lysis, failed to demonstrate significantdifferences among the cell types. Since maturation of erythroblasts to matured cells or erythrocytes isassociated with synthesis of band 3, hemoglobin, and spectrin and the loss of transferrin receptors, we concludethat none of these compounds serves as the specific receptor for staphylococcal a-toxin in BALB/c mice.

Staphylococcal a-toxin is an extracellular protein that isexcreted by most pathogenic strains of Staphylococcusaureus and is selectively hemolytic, dermonecrotic, andlethal for most laboratory animals (13, 17). The mechanismof hemolysis has been demonstrated with rabbit erythro-cytes to follow the following three-step sequence: binding ofa-toxin to specific receptors, formation of hexamer pores,which leads to leakage of small ions, and, finally, osmoticlysis of the cells (10, 11, 16, 21). The nature of the a-toxinreceptor on erythrocytes has not been definitely established,but there have been reports suggesting that the receptor is anN-acetylglucosamine-containing ganglioside (22), band 3glycoprotein (24), or glycophorin (6). Studies with murineerythroleukemia cell lines have demonstrated that matura-tion of mammalian erythrocytes from their nucleatederythroblast precursors involves preferential synthesis of thecell surface glycoprotein, band 3, and possibly glycophorin(14, 25, 28). Treatment of these cells with dimethyl sulfoxideinitiates the maturation process, in which not only are band3 (29) and glycophorin (14, 20) synthesized, which areunique to erythrocytes, but hemoglobin (18) and spectrin(14, 15) are synthesized as well. Additionally, maturation oferythroblasts leads to the loss of the transferrin receptor (27,30).We have been studying the maturation process of eryth-

rocytes by using splenic erythroblasts obtained from miceinfected with the anemia strain of Friend virus (M. J. Koury,S. T. Sawyer, and M. C. Bondurant, J. Cell. Physiol., inpress). In this system the maturation process is induced bythe addition of the natural hormone erythropoietin. Asexpected, erythroblast maturation involves induced synthe-sis of hemoglobin (Koury et al., in press), band 3, andspectrin and the loss of the transferrin receptor (unpublisheddata). An investigation of the timing of glycophorin synthe-sis in mouse cells is in progress. This murine erythropoietin-driven erythroblast maturation model, in which large num-bers of each cell type are available, provides an ideal systemto study a-toxin receptors. By comparing the susceptibilitiesof erythroblasts, erythropoietin-induced "matured" cells,and erythrocytes to ax-toxin, the role of band 3, spectrin, andthe transferrin receptor can be evaluated. Our results do notsupport the view that any of the molecules which are only

* Corresponding author.

synthesized late during erythrocyte differentiation can serveas the specific at-toxin receptor in murine erythrocytes.

MATERIALS AND METHODS

The at-toxin used was prepared by the pore glass method(12) and had a specific activity of 25,600 hemolytic units permg of protein. Hemolytic unit values were determined byusing rabbit erythrocytes as described by Bernheimer andSchwartz (7). Protein was measured by the method of Lowryet al. (23), using bovine serum albumin (Sigma Chemical Co.,St. Louis, Mo.) as the standard. 86RbCl (New England Nu-clear Corp., Boston, Mass.) had a specific activity of 3.91mCi/mg. The 86RbCl was used to measure the release ofsmall ions from the cells, and radioactivity was measured byadding the test sample (usually 10 ,u) to 10 ml of Aquasol andcounting the vials with a Beckman model LS-233 scintillationcounter.

Alpha-toxin induced release of 86Rb. To compare sensitiv-ity to a-toxin, erythroblasts, matured cells, and erythrocyteswere prelabeled with 86Rb. A 200-,u1 sample of each cell typesuspended in PBSA buffer (0.14 M NaCl, 0.01 M Na2HPO4,pH 7.4, 1 mg of bovine serum albumin per ml) was incubatedwith 0.2 mCi of 86RbCI for 30 min at 23°C. Typically, 1.5 x108 erythroblasts, 4 x 108 matured cells, and 3 x 108 adulterythrocytes were used. The cells were washed twice with30-ml volumes of PBSA buffer and were suspended in 800 ,ulof PBSA buffer. To confirm that each of the cell typesretained 86Rb, a 10-1±1 sample of each suspension wascounted and compared with a 10-,ul sample of the superna-tant obtained after centrifugation of a 50-11 portion with aBeckman Airfuge for 1.5 min.Hemolytic assays were done both by the hemoglobin

release procedure described by Bernheimer and Schwartz(7) and by a procedure involving continuous measurement ofthe reduction in turbidity. In this procedure the mouse cellsuspension was adjusted to an optical density at 660 nm of1.0 (approximately 0.065% adult BALB/c erythrocytes), andafter addition of a-toxin, the decrease in optical density wasfollowed over a 30-min period. With adult erythrocytes, thetwo methods gave comparable results and were equallysensitive.Female BALB/c mice that were 8 to 12 weeks old were

obtained from the National Institutes of Health, Bethesda,Md. Friend virus anemia-inducing strain FVA was pseudo-type SFFVA/FRE cl-3/MuLV(201) and was obtained from

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SUSCEPTIBILITY TO STAPHYLOCOCCAL ox-TOXIN 115

Be~

B

FIG. 1. Photomicrographs of Friend virus anemia strain-infectederythroblasts. (A) Spleen cells from BALB/c mice infected with theanemia strain of Friend virus that sedimented at 7 mm/h or greaterat unit gravity (erythroblasts). (B) Same cell population after 48 h ofculture with 0.2 U of erythropoietin per ml, (matured cells). Thearrow indicates an erythrocyte. Wright-stained, centrifuged prepa-ration. Bar = 20 p.m.

W. D. Hankins, National Institutes of Health. Virus was

maintained by passage of infectious plasma in BALB/c mice.Sheep plasma erythropoietin (4 U/mg of protein; step 3;Connaught Laboratories, Swiftwater, Pa.) was used forinduction of differentiation.

Isolation of splenic erythroblasts. Mice were injected viatail veins with approximately 104 spleen focus-forming units(2) of strain FVA. At 13 to 15 days after infection the micewere killed, and their spleens were removed. A single-cellsuspension of spleen cells in the Iscove modification ofDulbecco medium was made by passing the minced spleniccontents through nylon mesh bags taken from human bloodadministration sets (Fenwal Laboratories, Deerfield, Ill.).Cells from three to five spleens were pooled for eachexperiment. The spleen cells were separated by sedimenta-tion at 4°C for 4 h at unit gravity in a continuous gradient of1 to 2% deionized bovine serum albumin, as described byMiller and Phillips (26). The apparatus was a chamber with a

diameter of 180 mm (O. H. Johns Glass, Toronto, Ontario,Canada). Those cells sedimenting at velocities equal to orgreater than 7.0 mm/h were more than 90% erythroblasts(Koury et al., in press) and were used for culture.Erythroblast culture. Samples (15 ml) of splenic

erythroblasts were cultured in plastic plates (diameter, 100mm) at 37°C in humidified air containing 4% CO2. Themedium used was the Iscove modification of Dulbeccomedium supplemented with 0.8% methylcellulose, 30% fetalbovine serum, 1% deionized bovine serum albumin, 100 U ofpenicillin per ml, 100 p.g of streptomycin per ml, and 10-4 Ma-thioglycerol. The cultures contained 0.2 erythropoietinand 106 nucleated cells per ml of medium. Nucleated cellswere counted in a 0.1% aqueous solution of methylene blue

with a hemacytometer. After 48 h in culture with erythropoi-etin, the cells had synthesized hemoglobin and were in theterminal phases of maturation, as indicated by highly con-densed, eccentric nuclei and evidence of nuclear exclusionin a large fraction of the cells. In this report, we refer tothese erythropoietin-stimulated cells in the later stages ofterminal differentiation as matured cells.

RESULTSTreatment of BALB/c erythroblasts with 0.2 U of

erythropoietin for 2 days in culture induced several changesthat were observed morphologically (Fig. 1). In addition tosynthesis of hemoglobin, as evidenced by the developmentof a red color, the sizes of the cells were markedly reduced,resulting in a dramatic change in the ratio of cytoplasm tonucleus. Figure 1 also shows some matured cells in theprocess of extruding their nuclei, as well as a BALB/cerythrocyte for comparison.

Sensitivity to hemolysis of erythrocytes by a-toxin isspecies dependent; rabbit cells are among the most suscep-tible cells, and human cells are among the most resistant (1).This difference in sensitivity of erythrocytes to lysis bya-toxin is related to differences in the number of a-toxinreceptors on the erythrocyte membranes, as determined bya-toxin binding studies (3, 9-11). Mouse erythrocytes areapproximately one-tenth as sensitive as rabbit erythrocytes(4), and we confirmed this finding for BALB/c mouse periph-eral erythrocytes by using the hemoglobin release assay (7).BALB/c erythrocytes gave a value of 0.27 pug of a-toxin perml for the hemolytic unit, compared with a value of 0.04 pugof a-toxin per mnl for the hemolytic unit obtained with rabbiterythrocytes.The results of studies to measure the release of small ions

(Table 1) demonstrated that 86Rb was retained by all threecell preparations, indicating that the plasma membrane per-meability of the cells remained intact. The differences in theamount of radiolabel retained by each cell type reflect thedifferences in cell size and correlate well with the calculateddifferences in cytoplasm volume.The sensitivity to a-toxin of each cell type was measured

by testing the ability of a-toxin to induce the release of 86Rb.Two 300-,ul samples of each 86Rb-labeled cell suspensionwere taken; 30 of PBSA buffer was added to the firstsample (control), and 30 [L1 of a-toxin containing 0.8 mousehemolytic unit was added to the second sample. At 5-minintervals (up to 20 min), 40-,ul portions were removed, thecells were pelleted for 1 min with a Beckman Airfuge, and10-,ul samples of the clear supernatant were counted. Max-

TABLE 1. Labeling of BALB/c erythroblasts, Matured cells, anderythrocytes with "6RbCla

Radioactivity (cpm/10-,ul sample)Sample

Erythroblasts Matured cells Erythrocytes

First wash 8,847 7,993 7,853Second wash 2,871 1,257 204Cell supernatant 2,667 5,511 310Cell suspension 11,617 31,360 2,983

a Each cell type was incubated with 0.2 mCi of 86RbCI in 200 p.1 of PBSAbuffer at 23°C for 30 min. The cells were then washed with 30-mI volumes ofPBSA buffer and resuspended in 0.8 ml of the same buffer, and a 10-p.l sampleof the cell suspension was taken for counting. A 50-ld sample was centrifugedfor 1 min in a Beckman Airfuge, and a 10-pA sample of the supematant wascounted. The numbers of cells used in the initial labeling experiment were 1.8x 108, 5.3 x 108, and 3 x 108 cells for erythroblasts, matured cells, anderythrocytes, respectively. For further details see the text.

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116 HARSHMAN AND BONDURANT

imum release of 86Rb was determined by counting a sampleof cells after exposure to 1% Triton X-100. This maximumvalue agreed closely that the value obtained by counting thecell suspension before centrifugation. Our results correctedfor spontaneous release of radiolabel (control), are shown inFig. 2. Our data demonstrated that a-toxin induced therelease of 86Rb from each of the cell types. Furthermore, theerythroblasts, which did not have band 3 in their plasmamembranes, were as sensitive, if not more sensitive toa-toxin than the erythropoietin-treated matured cells, whichdid contain this glycoprotein on their surfaces. Similarresults were obtained in five different experiments withdifferent preparations of cells.

Lysis of BALB/c erythroblasts, matured cells, and erythro-cytes by a-toxin. To confirm that the sensitivities of the celltypes to a-toxin were correctly reflected by the 86Rb releaseassay, the sensitivities were tested by using a lytic assay.Each cell type was diluted in PI3SA buffer such that theoptical density at 660 nm was 1.0. To each cuvette 2.5 mousehemolytic units of a-toxin was added, and the reduction inturbidity (lysis) was recorded at 2-min intervals for 30 min.The resulting data (Fig. 3) support the conclusion thaterythroblasts and matured cells were equally sensitive toa-toxin-induced cell lysis. The apparent differences in extentof lysis (residual turbidity, measured at 30 min) were due tonuclei and were a reflection of the ratio of cytoplasm tonuclei for each cell type. When the extent of lysis at 30 minwas calculated as a percentage of the maximal value ob-tained after lysis of the plasma membranes with 1% TritonX-100, we obtained values of 97% for the erythroblasts, 91%for the matured cells, and 101% for the erythrocytes. Phase-contrast microscopy demonstrated that exposure to 1%Triton X-100 did not disrupt the nuclei of the erythroblastsor the erythropoietin-treated matured cells. Similar results

Erythrocytes100 _

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matured cells, and erythrocytes. Cells prelabeled with '6RbCl wereincubated with 2.5 mouse hemolytic units of a-toxin per ml. Atdifferent times samples were removed, and the cells were pelleted.Samples of the supernatant were counted to measure released 86Rb.Control cells, which were not treated with a-toxin, were used tomeasure spontaneous release. The data are expressed as percent-ages of 86Rb released after correction for sponltaneous (control)release. The average levels of spontaneous release for erythroblasts,matured cells, and erythrocytes were 36, 32, and 14%, respectively.For the concentrations of each cell type used see Table 1, footnotea. For further discussion see the text.

Ek 0.6_E0w0 0.5_0

0.4

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FIG. 3. Alpha-toxin-induced lysis oferythroblasts, matured cells,and erythrocytes. Samples of each cell type from the preparationsdescribed in Table 1, footnote a, were diluted to give an opticaldensity at 660 nm (OD 660 m,u) of 1.0. Alpha-toxin (2.5 mousehemolytic units) was added, and lysis of the cells was followed asthe reduction in optical density at 660 nm. The residual opticaldensity in the erythroblasts and matured cells was due to the nuclei,which were not lysed at the concentration of a-toxin used. Forfurther details see the text.

were obtained in four different experiments with differentpreparations of cells.

DISCUSSIONIt is the current view that a-toxin may interact with cells in

two distinct ways, which may be termed specific and non-specific. Specific interaction involves specific receptors andlow concentrations of a-toxin, shows high selectivity, and isexemplified by the hemolysis of rabbit-erythrocytes. Non-specific interaction does not involve specific receptors,requires 100 to 1,000 times the concentration of toxinrequired for specific reactions, shows little discrimination,represents penetration of lipid membranes by a putativehydrophobic region of the toxin, and is exemplified by lysisof human-erythrocytes and liposomes. The existence of twomechanisms of a-toxin interaction is supported by the fol-lowing lines of evidence. Binding studies of a-toxin andrabbit erythrocytes (3, 9-11) have demonstrated the pres-ence of a homogeneous, saturable receptor that specificallybinds a-toxin. Saturation of the receptors occurs at a-toxinconcentrations of 0.01 to 0.5 ,ug/ml, and the receptors arespecifically susceptible to degradation by pronase but not bytrypsin. In contrast to these studies, membranes from avariety of erythrocytes (5) and synthetic liposomes (17) thathave been exposed to 300 to 100 pLg of a-toxin per ml havebeen examined by electron mnicroscopy. Such membranesare coated with ringlike arrays which are identical to the 12Shexamer form of a-toxin. These hexamer forms have re-cently been isolated by Fussle et al. (19), who showed thatthe hexamers could be incorporated into liposomes andrepresented discrete transmembrane channels through whichsmall ions could pass. That a-toxin at high concentrations

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SUSCEPTIBILITY TO STAPHYLOCOCCAL a-TOXIN 117

can directly penetrate lipid monolayers has been demon-strated by using lipid films (8). The relationship of thehexamer forms to the "normal" action of a-toxin is notclear. First, ring forms are not observed on rabbit erythro-cyte membranes lysed at the usual hemolytic doses of toxin(0.01 to 0.1 ,ug/ml). Second, the 12S hexamer form of a-toxinis not itself hemolytic. Third, liposomes prepared with lipidsfrom human erythrocytes and rabbit erythrocytes show thesame high dose requirement for a-toxin (30 ,ug/ml) for lysis(13). Thus, direct interaction of a-toxin with membranelipids does not explain the more than 100-fold difference insusceptibility exhibited by intact human erythrocytes andrabbit erythrocytes. It is possible that the high-affinity re-ceptors present on rabbit erythrocytes but not detectable onhuman erythrocytes are themselves modified after bindingtoxin in such a way as to initiate cell damage or that theyfacilitate the formation of hexamers of a-toxin that permitleakage of small ions. The latter model views the specificreceptors as facilitators of hexamer formation.The exact nature of the a-toxin receptor remains elusive.

It has been reported to be an N-acetylglucosamine-contain-ing ganglioside (22), but Wiseman (31) has concluded thatthe reaction is probably nonspecific. Based on pronasesensitivity and interference of binding by lectins, Maharajand Fackrell (24) have suggested that erythrocyte band 3may be the specific receptor for a-toxin. Most recently,results of inhibition of hemolysis studies led Bernheimer andAvigad (6) to propose that glycophorin is the receptor. Todate, the direct isolation of an a-toxin receptor complex ofdefined composition has not been achieved.The availability of a model system to study erythroblast

maturation in vitro (Koury et al., in press) afforded anexceptional opportunity to critically evaluate the putativerole of band 3 as the a-toxin membrane receptor andpossibly glycophorin as well. Friend virus anemia strain-in-fected erythroblasts are nucleated progenitor cells of theerythropoietic pathway, which, like undifferentiated murineerythroleukemia cell lines (29), do not contain band 3 in theirmembranes (unpublished data). Culture of these cells witherythropoietin (Koury et al., in press) induces terminalmaturation and, of interest here, synthesis of band 3. Thus,if band 3 serves as the specific receptor for a-toxin, thesensitivities of these two cell types to a-toxin should bedifferent. The erythroblasts would be expected to be rela-tively resistant to a-toxin and, like human erythrocytes,which are devoid of a-toxin receptors, to require highconcentrations of a-toxin, characteristic of the nonspecificmode of a-toxin-cell interaction. In contrast, cells inducedto undergo terminal differentiation, matured cells, whichexpress band 3 on their surfaces, would be expected to be assensitive to a-toxin as the adult erythrocytes. When testedby both the release of 86Rb procedure (Fig. 2) and by theinduction of cell lysis method (Fig. 3), erythroblasts werefound to be as susceptible or more susceptible to a-toxinthan matured cells. Thus, our data do not support assign-ment of band 3 as the a-toxin receptor, at least not inBALB/c mouse erythrocytic cells. By extension, the knowninduction of spectrin (15) in matured cells and the loss oftransferrin receptors (27, 28) from differentiated erythro-blasts, events which we confirmed with our cells, by thesame reasoning, eliminate these two proteins as prospectivea-toxin receptors. Finally, if the result of glycophorin syn-thesis on human bone cells reported by Gahmberg et al. (20)is confirmed with the Friend virus anemia strain-infectederythroblasts used in this study, glycophorin also may beeliminated as a prospective a-toxin receptor.

ACKNOWLEDGMENTSWe thank Nancy Sugg for expert technical support and Karen

Perry for assisting in the preparation of the manuscript.This work was supported in part by grant PCM 8118653 from the

National Science Foundation.

LITERATURE CITED

1. Arbuthnott, J. P. 1970. Staphylococcal a-toxin, p. 189-232. InT. C. Montie, S. Kadis, and S. J. Ajl (ed.), Microbial toxins,vol. 3. Bacterial protein toxins. Academic Press, Inc., NewYork.

2. Axelrad, A. A., and R. A. Steeves. 1964. Assay for Friendleukemia virus: a rapid quantitative method based on enumer-ation of macroscopic spleen foci in mice. Virology 24:513-515.

3. Barei, G. M., and H. R. Fackrell. 1979. The binding of fluores-cene-labeled staphylococcal alpha toxin to erythrocytes. Can. J.Microbiol. 25:1219-1226.

4. Bernheimer, A. W. 1968. Cytolytic toxins of bacterial origin.Science 159:847-851.

5. Bernheimer, A. W. 1972. Hemolysis of streptococci: character-ization and effects on biological membranes, p. 19-31. InStreptococci and Streptococcal diseases. Academic Press, Inc.,New York.

6. Bernheimer, A. W., and L. S. Avigad. 1980. Inhibition ofbacterial and other cytolysins by glycophorin. FEMS Lett.9:15-17.

7. Bernheimer, A. W., and L. L. Schwartz. 1963. Isolation andcomposition of staphylococcal alpha toxin. J. Gen. Microbiol.30:455-468.

8. Buckelew, A. R., and G. Colacicco. 1971. Lipid monolayers:interactions with staphylococcal a-toxin. Biochim. Biophys.Acta 233:7-16.

9. Cassidy, P., and S. Harshman. 1976. lodination of a tyrosylresidue in staphylococcal a-toxin. Biochemistry 15:2342-2348.

10. Cassidy, P., and S. Harshman. 1976. Studies on the binding ofstaphylococcal 251I-labeled a-toxin to rabbit erythrocytes. Bio-chemistry 15:2348-2355.

11. Cassidy, P., and S. Harshman. 1976. Staphylococcal a-toxin.Properties of the iodotoxin derivative and its binding to eryth-rocytes, p. 707-720. In J. Jeljaszewicz (ed.), Staphylococci andstaphylococcal diseases. Fisher-Verlag, New York.

12. Cassidy, P., and S. Harshman. 1976. Purification and staphylo-coccal alpha toxin by adsorption chromatography on glass.Infect. Immun. 13:982-986.

13. Cassidy, P., H. R. Six, and S. Harshman. 1974. Biologicalproperties of staphylococcal a-toxin. Biochim. Biophys. Acta332:413-423.

14. Chang, H., P. J. Langer, and H. F. Lodish. 1976. Asynchronoussynthesis of erythrocyte membrane proteins. Proc. NatI. Acad.Sci. U.S.A. 73:3206-3210.

15. Eisen, H., R. Back, and R. Emery. 1977. Induction of spectrin inerythroleukemic cells transformed by Friend virus. Proc. NatI.Acad. Sci. U.S.A. 74:3898-3902.

16. Freer, J. H., and J. P. Arbuthnott. 1983. Toxins of Staphylo-coccus aureus. Pharm. Ther. 19:55-106.

17. Freer, J. H., J. P. Arbuthnott, and A. S. Bernheimer. 1968.Interaction of staphylococcal a-toxin with artificial and naturalmembranes. J. Bacteriol. 95:1153-1168.

18. Friend, C., W. Scher, J. G. Holland, and T. Sato. 1971.Hemoglobin synthesis in murine virus-induced leukemic cells invitro: stimulation of erythroid differentiation by dimethyl sulf-oxide. Proc. Natl. Acad. Sci. U.S.A. 68:378-382.

19. Fussle, R., S. Bhakdi, A. Sziegoliet, J. Tranum-Jensen, T. Kranz,and H.-J. Wellensiek. 1981. On the mechanism of membranedamage by Staphylococcus aureus a-toxin. J. Cell Biol.91:85-94.

20. Gahmberg, C. G., M. Tokinen, and L. C. Anderson. 1978.Expression of the major sialoglycoprotein (glycophorin) inerythroid cells in human bone marrow. Blood 52:379-387.

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23. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

24. Maharaj, I., and H. B. Fackrell. 1980. Rabbit erythrocyte band3: a receptor for staphylococcal alpha toxin. Can. J. Microbiol.26:524-531.

25. Marks, P. A., and R. A. Rifkind. 1978. Erythroleukemic differ-entiation. Annu. Rev. Biochem. 47:419-448.

26. Miller, R. G., and R. A. Phillips. 1969. Separation of cells byvelocity sedimentation. J. Cell Physiol. 73:832-833.

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