Experimental Cell Research 93 (1975) 235-239
UNIQUE UTILIZING
TECHNIQUES MITHRAMYCIN
FOR CELL
CYCLE
AND
MICROFLUOROMETRY
FLOW
ANALYSIS
R. A. TOBEY’ and H. A. CRISSMAN2 ‘Cellular
and Molecular Radiobiology Group and 2Biophysics and Instrumentation Los Alamos Scientific Laboratory, University of California, Los Alamos, NM 87544, USA
Group,
SUMMARY Cell cycle analysis through utilization of the mithramycinlflow microfluorometric technique combines simplicity (one-step cell preparation) with speed (results available within 20 mitt after removal of a sample from a culture). Furthermore, the technique is useful in many situations in which standard 3H-thymidine autoradiography and mitotic accumulation are unsatisfactory. Examples are provided which demonstrate a continuous monitoring of experiments in progress, including analysis of populations devoid of cells in S or M phases, analysis of arrested or slowly progressing populations resulting from exposure to toxic agents, detection of abnormalities in mitosis such as nondisjunction and polyploidization, and localization within the cell cycle of dying cells in cultures exposed to toxic agents.
Studies of cell cycle specific biochemical scribed previously [5]. Cells to be stained with mithramycin were centrifuged and resuspended in 25% events in synchronized cultures [l] or aqueous ethanol containing 100 pg/ml of mithramycin kinetic responses to drug treatment [2] re- and I5 mM MgC&; after 15-20 min, the cells in mithramycin-containing solution were examined diquire precise localization of the population rectly in the Los Alamos FMF, utilizing a wavelength within the cell cycle. In populations such setting of 457 nm [6]. 1,34G(2-Chloroethyl)-1-nitrosourea (BCNU), NSC as these, cells frequently are arrested in no. 409962, and chlorozotocin, NSC no. 178248,were specific portions of the cell cycle or traverse obtained through Drug Research and Development, Division of Cancer Treatment, National Cancer Instithe cycle at a grossly reduced rate so that tute. Supplies of mithmmycin were obtained both from standard analytical techniques are unsatis- the National Cancer Institute and as a gift, generously provided by Pfizer Laboratories Division, New York factory. Consequently, we have developed City, N.Y., USA. a technique, involving the Los Alamos flow microfluorometer (FMF) [3] in conjunction RESULTS with mithramycin [4], which permits detailed cell cycle analysis of both normal Analysis by FMF permits determination of and abnormal populations. the phase in the cell cycle in which a cell resides based upon DNA content (i.e., a cell in G2 or M contains twice as much MATERIALS AND METHODS DNA as a G 1 cell, etc.). Cells are treated Line CHO Chinese hamster cells were mown in suspension culture in F-IO medium supplemented with with a fluorescent dye which binds specifi10% calf serum and 5% fetal calf serum. In certain experiments, cells were synchronized in G 1 arrest by cally to DNA; following laser excitation, maintenance in isoleucine-deficient medium, as de- the cells fluoresce with an intensity proExprl Cell Res 93 (1975)
236
Tohey and Crissman
DNA content. Cells in S phase are located at intermediate positions between the peaks. Also included in fig. IA is the fraction of cells in each phase of the cell cycle, obtained by computer fit of the fluorescence distribution patterns [8]. In all experiments, the results obtained through FMF analysis and by standard cell cycle analytical techniques were in excellent agreement, as documented previously [8]. The principal disadvantages of the acriflavine-Feulgen procedure are (a) the complexity (large number) of operations required for dispersal, fixation, and staining; (b) the harsh acid hydrolysis treatment during staining of the cells; and (c) the duration of the fixation period (12-18 h for optimum results). To overcome these difficulties, we have devised a technique employing the drug mithramycin in FMF analysis [6]. Cell cycle analysis of the same exponentially growing culture of CHO cells shown in fig. 1A with the mithramycin technique yielded the results shown in fig. 1b. The mithramycin-generated results are essentially identical to those obtained with the acriflavine procedure, yet the three major disadvantages of the acriflavine protocol have been circumvented through Fig. I. Abscissa: rel. DNA content; or&are: no. of use of the mithramycin technique. cells (X 10-Z). DNA distribution obtained by FMF analysis of exIn view of the rapidity with which results nonentiallv growing cultures of line CHO Chinese are obtained utilizing the mithramycin/FMF hamster celk stained with either (A) acriflavineFeulgen procedure [7] or(B) mithramycin protocol [6]. technique, it is now possible to continuousThe percentages of cells in G I, S, and GZ+M were obtained by computer-fit analyses of the DNA dis- ly monitor population kinetics in ongoing ttibution c&ves [8] experiments. For example, in fig. 2 are presented results obtained in an experiportional to DNA content. In fig. 1A, our ment designed to detect the kinetic restandard acriflavine-Feulgen procedure [7] sponse of initially G l-arrested CHO cells was utilized to obtain the population DNA treated for 2 h with the anticancer drug distribution from an exponentially growing chlorozotocin, then returned to cycle in culture. The peak at a scale value of ap- drug-free medium. The results in fig. 2 inprox. 3 1 represents cells in G 1, while the dicate that chlorozotocin (a) did not affect second peak at a scale value of 62 repre- the rate of progression through G 1; (6) insents cells in G2 and M, with twice the duced a large increase in the time required Exptr Ceil Res 93 (1975)
Cell cycle analysis via mithramycinlFMF
40 20
237
arrested in G 1, or in S phase under conditions where thymidine transport is impaired or inhibited [9], are readily analysable by FMF analytical techniques. A difficult parameter to assess in drugtreated or lytic virus-infected cultures is the phase (or phases) of the cell cycle in which
v .-**o ~: , ,,,, I”““““” n O
cells
are dying.
The acriflavine-Feulgen
protocol involves treatment of cells with EDTA, trypsin, and acid hydrolysis [7] 0 procedures which destroy dying and damaged cells, selectively removing them from 80 80 the population [lo]. Since the mithramycin technique eliminates the manipulations in 60 60 the standard protocol that destroy fragile 40 40 cells, duplicate aliquots of an experimental culture may be stained with either acri20 20 flavine or mithramycin and an attempt may be made to detect the differences reflecting 0 0 cell loss in the Feulgen procedure. In fig. 3 24 0 0 0 8 16 16 24 Fig. 2. Abscissa: time after reversal of G 1 arrest are results from an exponential population (hours); ordinate: % of cells (A) G 1; (B) S; (C) G 2; (0) of CHO cells treated for 2 h with the chemodivided. Progression of synchronized CHO cells through the therapeutic agent BCNU, followed by incell cvcle followine treatmentwith the chemotheraueu- cubation for 96 h in drug-free medium prior tic agknt chlorozokcin. Cells arrestedin G 1 by cultivation in isoleucine-deficient medium for 36 h TS. 91 to FMF analysis. The intact cell population were treated for a 2-h period with 8 pgl& o? is represented by open circles, while the chlorozotocin (survival value of approx. 5%), then intact plus fragile population is indicated returned to cycle by resuspension in fresh, complete, drug-free medium (0). A nondrug-treated culture by solid figures. The results are most readiserved as control (0). Samples wereremoved at intervals for determination of the fraction of cells in each ly interpreted by consideration of the relaphase of the cell cycle by the mithramycin/FMF technique. The divided fraction represents N/Nrl so that tive ratios of 2C (G l), 4C (G 2 and M), and a true population doubling would appear as an increase 8C (DNA-containing cells in the cultures from 0 to 1 on the scale provided. stained by the two procedures. The 4C and 8C DNA-containing cells were reduced for progression through S phase; (c) caused proportionately to the greatest extent in the most of the cells in the population ultimale- Feulgen-stained cells, indicating that these ly to accumulate in the G2 phase of the cells are the most sensitive to disruption cell cycle; and (d) allowed only a small and, therefore, are among the first cells to fraction of the population to divide. Note die in BCNU-treated populations. that the arrested populations such as those The FMF data in fig. 3 yield additional appearing at late times after treatment with information which is difficult to obtain by chlorozotocin were readily analyzable by other techniques. There was a pronounced FMF analysis, although it would be difficult broadening of the base of the 2C (G 1) or impossible to locate these cells utilizing peak in the BCNU-treated culture which standard cell cycle analysis. Similarly, cells was never observed in untreated cultures. ExptlCeNRes 93(1975)
238
Tobey and Crissman
3. Abscissa: rel. DNA content; ordinate: no. of cells. Detection of DNA distributions in populations of intact cells and in populations of intact plus fragile cells in a culture treated with the chemotherapeutic compound BCNU. Exponentially growing cultures of CHO cells were treated for 2 h with 10 &ml of BCNU (survival value of approx. 1% [2]), and then the cells were resuspended in drug-free, complete medium. After-96 h, FMF analysis was performed utilizing the standard acriflavine-Feulgen procedure [7] to yield the intact cell DNA oooulation distribution (0) or the mithramycin protocol ‘[6] to yield the DNA population distribution for the intact ~1~sfragile cell oooulation (0). Approx. 2OOClOO cells were examined in each preparation.
Fig.
This broadening reflects nondisjunctive errors occurring during mitosis, resulting in an unequal partitioning of DNA between daughter cells, as documented previously [ll]. Furthermore, BCNU induced the formation of polyploid cells (cells with an 8C DNA content). Thus, in addition to its other advantages, the mithramycin-FMF procedure may be utilized to detect abnormalities in progression through mitosis. DISCUSSION In this report, we have presented several examples of the versatility of the mithramycin-FMF technique. Our experience inExprl Cell Res 93 (197.5)
dicates that, for maximum analytical detail, standard cell cycle techniques should be combined with the mithramycin protocol [9]. Although the data presented in this report have been obtained with CHO cells, the mithramycin technique works equally well with a wide variety of cell types and for cells grown in either monolayer or suspension culture. For example, excellent results have been obtained with human W138, HeLa, and FL cells, L-929 mouse cells, EMT-6 mouse transplantable tumor cells, bovine and human lymphocytes, and human squamous cells from cervical samples. In fact, all cell types thus far examined by the mithramycin technique have yielded reproducible DNA distributions. Although we have utilized the Los Alamos FMF in these studies, we would expect that any device with a source of sufficient intensity at 395 nm, the excitation maximum for mithramycin bound to cells [6], should prove satisfactory for measurements of this nature. In particular, either scanning or flow systems employing lasers, mercury vapor lamps, etc., as excitation sources may be ideally suited for analysis of mithramycin-stained cells. Among advantages of the mithramycim FMF procedure are the ability to (a) monitor population kinetics in ongoing experiments, with the added option of altering an experiment in progress in response to a population change; (b) localize cells within S phase, distinguishing between early, mid, and late S phase, etc.; (c) analyze populations which have been prelabeled with radiocompounds under conditions where 3H-thymidine autoradiography is impossible; (d) monitor populations comprised of slowly progressing or arrested cells; (e) analyze populations devoid of cells in S or M; v) analyse populations containing cells unable to transport 3H-thy-
Cell cycle analysis via mithramycinlFMF midine; (g) detect abnormalities in progression through mitosis such as nondisjunction or polyploidization; and (h) distinguish between intact and fragile (dying) cells in drug-treated or virus-infected cells. The excellent technical assistance of Mrs Phyllis C. Sanders and Mr Melvin S. Oka is gratefully acknowledged. This study was supported by contract NIH-CR-(71)-56 from the Division of Cancer Treatment, NCI, NIH, Department of Health, Education, and Welfare, Bethesda, Md, under interagency agreement with the US AEC.
REFERENCES I. Tobey, R A, Gurley, L R, Hildebrand, C E, Ratliff, R L & Walters, R A. Control of oroliferation in animals cells (ed B Clarkson & R-Baserga) p. 665. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1974).
16-751809
239
2. Tobey, R A & Crissman, H A, Cancer res 935 (1975) 460. 3. Holm, D M & Cram, L. S, Exptl cell res 80 (1973) 105. 4. Ward, DC, Reich, E&Goldberg, I H, Science 149 (1965) 1259. 5. Tobey, R A & Ley, K D, Cancer res 31 (1971) 46. 6. Crissman, H A & Tobev, R A, Science 184 (1974) 1297. 7. Tobey, R A, Crissman, H A & Kraemer, P M, J cell biol54 (1972) 638. 8. Dean, P N & Jett, J H, J cell biol60 (1974) 523. 9. Tobey, R A, Methods in cell biology (ed D M Prescott) vol. 6, p. 67. Academic Press, New York (1973). 10. DeLuca, C, Exptl cell res 40 (1965) 186. 11. Kraemer, P M, Deaven, L L, Crissman, H A & Van Dilla, M A, Advances in cell and molecular biology (ed E J DuPraw) vol. 2, p. 47. Academic Press, New York (1972). Received November 20, 1974 Revised version received December 27, 1974
Exptl Cell Res 93 (1975)
UNIQUE UTILIZING
TECHNIQUES MITHRAMYCIN
FOR CELL
CYCLE
AND
MICROFLUOROMETRY
FLOW
ANALYSIS
R. A. TOBEY’ and H. A. CRISSMAN2 ‘Cellular
and Molecular Radiobiology Group and 2Biophysics and Instrumentation Los Alamos Scientific Laboratory, University of California, Los Alamos, NM 87544, USA
Group,
SUMMARY Cell cycle analysis through utilization of the mithramycinlflow microfluorometric technique combines simplicity (one-step cell preparation) with speed (results available within 20 mitt after removal of a sample from a culture). Furthermore, the technique is useful in many situations in which standard 3H-thymidine autoradiography and mitotic accumulation are unsatisfactory. Examples are provided which demonstrate a continuous monitoring of experiments in progress, including analysis of populations devoid of cells in S or M phases, analysis of arrested or slowly progressing populations resulting from exposure to toxic agents, detection of abnormalities in mitosis such as nondisjunction and polyploidization, and localization within the cell cycle of dying cells in cultures exposed to toxic agents.
Studies of cell cycle specific biochemical scribed previously [5]. Cells to be stained with mithramycin were centrifuged and resuspended in 25% events in synchronized cultures [l] or aqueous ethanol containing 100 pg/ml of mithramycin kinetic responses to drug treatment [2] re- and I5 mM MgC&; after 15-20 min, the cells in mithramycin-containing solution were examined diquire precise localization of the population rectly in the Los Alamos FMF, utilizing a wavelength within the cell cycle. In populations such setting of 457 nm [6]. 1,34G(2-Chloroethyl)-1-nitrosourea (BCNU), NSC as these, cells frequently are arrested in no. 409962, and chlorozotocin, NSC no. 178248,were specific portions of the cell cycle or traverse obtained through Drug Research and Development, Division of Cancer Treatment, National Cancer Instithe cycle at a grossly reduced rate so that tute. Supplies of mithmmycin were obtained both from standard analytical techniques are unsatis- the National Cancer Institute and as a gift, generously provided by Pfizer Laboratories Division, New York factory. Consequently, we have developed City, N.Y., USA. a technique, involving the Los Alamos flow microfluorometer (FMF) [3] in conjunction RESULTS with mithramycin [4], which permits detailed cell cycle analysis of both normal Analysis by FMF permits determination of and abnormal populations. the phase in the cell cycle in which a cell resides based upon DNA content (i.e., a cell in G2 or M contains twice as much MATERIALS AND METHODS DNA as a G 1 cell, etc.). Cells are treated Line CHO Chinese hamster cells were mown in suspension culture in F-IO medium supplemented with with a fluorescent dye which binds specifi10% calf serum and 5% fetal calf serum. In certain experiments, cells were synchronized in G 1 arrest by cally to DNA; following laser excitation, maintenance in isoleucine-deficient medium, as de- the cells fluoresce with an intensity proExprl Cell Res 93 (1975)
236
Tohey and Crissman
DNA content. Cells in S phase are located at intermediate positions between the peaks. Also included in fig. IA is the fraction of cells in each phase of the cell cycle, obtained by computer fit of the fluorescence distribution patterns [8]. In all experiments, the results obtained through FMF analysis and by standard cell cycle analytical techniques were in excellent agreement, as documented previously [8]. The principal disadvantages of the acriflavine-Feulgen procedure are (a) the complexity (large number) of operations required for dispersal, fixation, and staining; (b) the harsh acid hydrolysis treatment during staining of the cells; and (c) the duration of the fixation period (12-18 h for optimum results). To overcome these difficulties, we have devised a technique employing the drug mithramycin in FMF analysis [6]. Cell cycle analysis of the same exponentially growing culture of CHO cells shown in fig. 1A with the mithramycin technique yielded the results shown in fig. 1b. The mithramycin-generated results are essentially identical to those obtained with the acriflavine procedure, yet the three major disadvantages of the acriflavine protocol have been circumvented through Fig. I. Abscissa: rel. DNA content; or&are: no. of use of the mithramycin technique. cells (X 10-Z). DNA distribution obtained by FMF analysis of exIn view of the rapidity with which results nonentiallv growing cultures of line CHO Chinese are obtained utilizing the mithramycin/FMF hamster celk stained with either (A) acriflavineFeulgen procedure [7] or(B) mithramycin protocol [6]. technique, it is now possible to continuousThe percentages of cells in G I, S, and GZ+M were obtained by computer-fit analyses of the DNA dis- ly monitor population kinetics in ongoing ttibution c&ves [8] experiments. For example, in fig. 2 are presented results obtained in an experiportional to DNA content. In fig. 1A, our ment designed to detect the kinetic restandard acriflavine-Feulgen procedure [7] sponse of initially G l-arrested CHO cells was utilized to obtain the population DNA treated for 2 h with the anticancer drug distribution from an exponentially growing chlorozotocin, then returned to cycle in culture. The peak at a scale value of ap- drug-free medium. The results in fig. 2 inprox. 3 1 represents cells in G 1, while the dicate that chlorozotocin (a) did not affect second peak at a scale value of 62 repre- the rate of progression through G 1; (6) insents cells in G2 and M, with twice the duced a large increase in the time required Exptr Ceil Res 93 (1975)
Cell cycle analysis via mithramycinlFMF
40 20
237
arrested in G 1, or in S phase under conditions where thymidine transport is impaired or inhibited [9], are readily analysable by FMF analytical techniques. A difficult parameter to assess in drugtreated or lytic virus-infected cultures is the phase (or phases) of the cell cycle in which
v .-**o ~: , ,,,, I”““““” n O
cells
are dying.
The acriflavine-Feulgen
protocol involves treatment of cells with EDTA, trypsin, and acid hydrolysis [7] 0 procedures which destroy dying and damaged cells, selectively removing them from 80 80 the population [lo]. Since the mithramycin technique eliminates the manipulations in 60 60 the standard protocol that destroy fragile 40 40 cells, duplicate aliquots of an experimental culture may be stained with either acri20 20 flavine or mithramycin and an attempt may be made to detect the differences reflecting 0 0 cell loss in the Feulgen procedure. In fig. 3 24 0 0 0 8 16 16 24 Fig. 2. Abscissa: time after reversal of G 1 arrest are results from an exponential population (hours); ordinate: % of cells (A) G 1; (B) S; (C) G 2; (0) of CHO cells treated for 2 h with the chemodivided. Progression of synchronized CHO cells through the therapeutic agent BCNU, followed by incell cvcle followine treatmentwith the chemotheraueu- cubation for 96 h in drug-free medium prior tic agknt chlorozokcin. Cells arrestedin G 1 by cultivation in isoleucine-deficient medium for 36 h TS. 91 to FMF analysis. The intact cell population were treated for a 2-h period with 8 pgl& o? is represented by open circles, while the chlorozotocin (survival value of approx. 5%), then intact plus fragile population is indicated returned to cycle by resuspension in fresh, complete, drug-free medium (0). A nondrug-treated culture by solid figures. The results are most readiserved as control (0). Samples wereremoved at intervals for determination of the fraction of cells in each ly interpreted by consideration of the relaphase of the cell cycle by the mithramycin/FMF technique. The divided fraction represents N/Nrl so that tive ratios of 2C (G l), 4C (G 2 and M), and a true population doubling would appear as an increase 8C (DNA-containing cells in the cultures from 0 to 1 on the scale provided. stained by the two procedures. The 4C and 8C DNA-containing cells were reduced for progression through S phase; (c) caused proportionately to the greatest extent in the most of the cells in the population ultimale- Feulgen-stained cells, indicating that these ly to accumulate in the G2 phase of the cells are the most sensitive to disruption cell cycle; and (d) allowed only a small and, therefore, are among the first cells to fraction of the population to divide. Note die in BCNU-treated populations. that the arrested populations such as those The FMF data in fig. 3 yield additional appearing at late times after treatment with information which is difficult to obtain by chlorozotocin were readily analyzable by other techniques. There was a pronounced FMF analysis, although it would be difficult broadening of the base of the 2C (G 1) or impossible to locate these cells utilizing peak in the BCNU-treated culture which standard cell cycle analysis. Similarly, cells was never observed in untreated cultures. ExptlCeNRes 93(1975)
238
Tobey and Crissman
3. Abscissa: rel. DNA content; ordinate: no. of cells. Detection of DNA distributions in populations of intact cells and in populations of intact plus fragile cells in a culture treated with the chemotherapeutic compound BCNU. Exponentially growing cultures of CHO cells were treated for 2 h with 10 &ml of BCNU (survival value of approx. 1% [2]), and then the cells were resuspended in drug-free, complete medium. After-96 h, FMF analysis was performed utilizing the standard acriflavine-Feulgen procedure [7] to yield the intact cell DNA oooulation distribution (0) or the mithramycin protocol ‘[6] to yield the DNA population distribution for the intact ~1~sfragile cell oooulation (0). Approx. 2OOClOO cells were examined in each preparation.
Fig.
This broadening reflects nondisjunctive errors occurring during mitosis, resulting in an unequal partitioning of DNA between daughter cells, as documented previously [ll]. Furthermore, BCNU induced the formation of polyploid cells (cells with an 8C DNA content). Thus, in addition to its other advantages, the mithramycin-FMF procedure may be utilized to detect abnormalities in progression through mitosis. DISCUSSION In this report, we have presented several examples of the versatility of the mithramycin-FMF technique. Our experience inExprl Cell Res 93 (197.5)
dicates that, for maximum analytical detail, standard cell cycle techniques should be combined with the mithramycin protocol [9]. Although the data presented in this report have been obtained with CHO cells, the mithramycin technique works equally well with a wide variety of cell types and for cells grown in either monolayer or suspension culture. For example, excellent results have been obtained with human W138, HeLa, and FL cells, L-929 mouse cells, EMT-6 mouse transplantable tumor cells, bovine and human lymphocytes, and human squamous cells from cervical samples. In fact, all cell types thus far examined by the mithramycin technique have yielded reproducible DNA distributions. Although we have utilized the Los Alamos FMF in these studies, we would expect that any device with a source of sufficient intensity at 395 nm, the excitation maximum for mithramycin bound to cells [6], should prove satisfactory for measurements of this nature. In particular, either scanning or flow systems employing lasers, mercury vapor lamps, etc., as excitation sources may be ideally suited for analysis of mithramycin-stained cells. Among advantages of the mithramycim FMF procedure are the ability to (a) monitor population kinetics in ongoing experiments, with the added option of altering an experiment in progress in response to a population change; (b) localize cells within S phase, distinguishing between early, mid, and late S phase, etc.; (c) analyze populations which have been prelabeled with radiocompounds under conditions where 3H-thymidine autoradiography is impossible; (d) monitor populations comprised of slowly progressing or arrested cells; (e) analyze populations devoid of cells in S or M; v) analyse populations containing cells unable to transport 3H-thy-
Cell cycle analysis via mithramycinlFMF midine; (g) detect abnormalities in progression through mitosis such as nondisjunction or polyploidization; and (h) distinguish between intact and fragile (dying) cells in drug-treated or virus-infected cells. The excellent technical assistance of Mrs Phyllis C. Sanders and Mr Melvin S. Oka is gratefully acknowledged. This study was supported by contract NIH-CR-(71)-56 from the Division of Cancer Treatment, NCI, NIH, Department of Health, Education, and Welfare, Bethesda, Md, under interagency agreement with the US AEC.
REFERENCES I. Tobey, R A, Gurley, L R, Hildebrand, C E, Ratliff, R L & Walters, R A. Control of oroliferation in animals cells (ed B Clarkson & R-Baserga) p. 665. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1974).
16-751809
239
2. Tobey, R A & Crissman, H A, Cancer res 935 (1975) 460. 3. Holm, D M & Cram, L. S, Exptl cell res 80 (1973) 105. 4. Ward, DC, Reich, E&Goldberg, I H, Science 149 (1965) 1259. 5. Tobey, R A & Ley, K D, Cancer res 31 (1971) 46. 6. Crissman, H A & Tobev, R A, Science 184 (1974) 1297. 7. Tobey, R A, Crissman, H A & Kraemer, P M, J cell biol54 (1972) 638. 8. Dean, P N & Jett, J H, J cell biol60 (1974) 523. 9. Tobey, R A, Methods in cell biology (ed D M Prescott) vol. 6, p. 67. Academic Press, New York (1973). 10. DeLuca, C, Exptl cell res 40 (1965) 186. 11. Kraemer, P M, Deaven, L L, Crissman, H A & Van Dilla, M A, Advances in cell and molecular biology (ed E J DuPraw) vol. 2, p. 47. Academic Press, New York (1972). Received November 20, 1974 Revised version received December 27, 1974
Exptl Cell Res 93 (1975)
