Cytotechnology 8: 65-74, 1992. 9 1992Khtwer Academic Publishers. Printed in tile Netherlands.

The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures Jos6 M. Coco-Martin, Jan W. Oberink, Tiny A.M. van der Velden-de Groot and E. Coen Beuvery Department o f Inactivated Viral Vaccines, National hlstitute o f Public Health and Environmental Protection (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands Received 14 October 1991; accepted in revised form I April 1992

Key words: flow cytometry, hybridomas, monoclonal antibodies, cytoplasmic IgG, membrane IgG, cell

cycle distribution Abstract

Flow cytometric (FC) analysis was applied to determine changes at cellular level during the cultivation of hybridoma cell line MN12 in a suspension batch culture. The relative cell size, cytoplasmic and membrane IgG content and the viability were monitored. Besides, the specificity of the cytoplasmic and membrane IgG was ascertained by means of a synthetic peptide containing the antigenic epitope recognized by the antibody. Cell size was found to increase during the exponential growth phase. The viability as determined by FC follows a similar pattern with the viability data obtained by the conventional trypan blue exclusion test. The relative cytoplasmic and membrane IgG contents were high during the exponential growth and low during stationary phase. Measurement of cell cycle distribution and the antibody content in the culture fluid, indicated that the major part of the cytoplasmic IgG is secreted by cells in the Gl-phase. It is concluded that flow cytometry is a useful tool to characterize hybridoma cell lines in a suspension batch culture.

Introduction

The introduction of flow cytometry (FC), has facilitated the use of fluorochrome-conjugated antibodies for the characterization of mammalian cells (Aubry et al., 1990; Terstappen et al., 1989) and bacteria. FC measurement allows a rapid analysis of cell populations and large numbers of cell samples can be quantitatively analyzed. Initially, FC has had a significant impact on the study of cell populations of the immune system, such as B-cells (Hoven et al., 1989) or T-lymphocytes (Festin et al., 1987; Festin et al., 1990). FC is not only an excellent tool to determine relative

amounts of cell surface antigens (Wing et al., 1990) but is also suitable to determine cytoplasmic antigens (Rigg et al., 1989) and nuclear components (Kurki et al., 1988; Clevenger et al., 1985). Jacobberger et al. (1986) described the quantification of the SV40 T-antigen in the cytoplasm of SV40 transformed and lyrically infected cells. Several authors have described the use of FC to measure the relative DNA content of cells or to follow the cell cycle, after labelling of the Cel,ls with propidium iodide (PI) (Rabinovitch et aL, 1988; Dolbeare etal., 1983; Dean etal., 1982). PI is a fluorescent DNA-intercalating agent which,

66 after permeabilization of the cells with ethanol or methanol is able to cross the cell membrane. 5-Bromodesoxyuridine (BrdU), a thymidine analogue, in combination with anti-BrdU monoclonal antibodies is also widely used to determine cellular proliferation. Another application of flow cytometry is the measurement of intracellular pH (Hedley et al., 1989; Rink et al., 1982). During the last few years flow cytometry has entered the field of biotechnology, e.g., for selection of isotype switch variants of hybridoma cells (Dangl and Herzenberg, 1982) or quadromas (Koolwijk et al., 1988) and as an analytical tool during large-scale cultivation of mammalian cells used for the production of biologicals (K16ppinger et al., 1991). In the present study the potential of FC was investigated to determine cellular properties of the hybridoma cell line M N I 2 in suspension batch culture. The relative amount of cytoplasmic and surface IgG was determined. The specificity of the surface and cytoplasmic IgG of the MN12 cell line was studied using a fluorescein labelled peptide which has the amino acid sequence corresponding to that of the epitope recognized by MNI2. Moreover, FC was used to analyze the cell cycle and to determine viability of the cells.

Materials and methods

Cultivation of hybridoma cell line MN12 Hybridoma cell line MN12 (IgG2a,k; specificity: outer membrane protein P1.16 of Neisseria meningitidis; kindly provided by Dr. J.T. Poolman, RIVM, Bilthoven, The Netherlands), was grown in Iscove's modified Dulbecco's medium (Gibco Laboratories, Grand Island, NY) supplemented with 0.25% (w/v) Primatone RL (Sheffield Products, Norwich, NY), 5% (v/v) heat-inactivated (56~ for 30 min) fetal bovine serum (FBS) (Flow Laboratories, Woodcock Hill, UK), and antibiotics (35,000 U/I polymyxin B, 14,000 U/1 neomycin, and 75,000 U/1 streptomycin). The cells were cultivated in I000 ml spinner-flasks (working volume: 500 ml) during five days using

a stirrer speed of 50 rpm. Samples were collected daily for measurement of antibody concentration, determination of viability and FC analysis. Cell number and viability Cell number and viability were determined by trypan blue exclusion (Patterson, 1979). Mouse IgG specific ELISA Wells of polystyrene microtiter plates (Flow Laboratories, Woodcock Hill, UK) were coated overnight at room temperature with sheep anti-mouse IgG (SMUG 73; 28 kt~ml in PBS, pH 7.3; RIVM). The plates were washed three times with PBS, containing 0.05% Tween 20 (Merck, Darmstadt, FRG) (PBS-Tween). Next, samples were added, prediluted in PBS-Tween containing 0.5% BSA (PBS-Tween-BSA) in eight twofold dilution steps (100 I.tl/well). After 1 h at 37~ the wells were washed three times with PBS-Tween. Horseradish peroxidase (HRPO)-labelled anti-mouse IgG, Fc specific (Cooper Biomedical, Malvem, PA) was added (100 ~tl 1:2000 diluted in PBS-TweenBSA) to each well. After incubation for 1 h at 37~ the wells were washed three times with PBS-Tween and 100 btl of the enzyme substrate, 300 mg 3,3',5,5'-tetramethylbenzidine (Sigma, St. Louis, MO) in 50 ml dimethyl sulphoxide (Merck, Darmstadt, FRG), was added. After incubation for 3-7 min, 2 M H2SO4 was added (100 lal/well) and the absorbance was measured at 450 nm with a microtiter plate reader (Flow Laboratories, Woodcock Hill, UK). Peptide synthesis Monoclonal antibody (Mab) MN12 recognizes a linear epitope containing the amino acid sequence Tyr-Tyr-Thr-Lys-Asp-Thr-Asn-Asn-Asn-Leu of the outer membrane protein PI.16 of Neisseria Meningitides (McGuinness et al., 1990). This peptide was synthesized and labelled with 5(iodoacetamido)fluorescein as describedtby Drijfhout et al., (1990).

Flow cytometric analysis Samples were analyzed with a flow cytometer (FACScan; Becton Dickinson BV, Etten-Leur, The Netherlands). An HP310 computer (HewlettPackard Corporation, Pittsburgh, PA) with a Consort 30 program (Becton Dickinson) was used for data processing~ Dead cells were gated out according to their forward-angle and right-angle scattering properties.

Viability measurement. Scatter properties, forward scatter (FSC) and side scatter (SSC), were measured on a flow cytometer without pretreatment of the samples. Cytoplasmic lgG content. Cell suspensions of the MN12 cell line (106 cells/ml) were washed three times with cold (4~ PBS, pH 7.3. After the final washing and resuspension, the cells were fixed and permeabilized by adding 1 ml methanol (Merck, Darmstadt, FRG) at - 7 0 ~ while gently stirring. Fixed cells were kept at - 2 0 ~ for at least 15 h, and were washed three times with PBS, pH 7.3 containing 0.5% BSA (PBS-BSA). The cells were incubated with fluorescein isothiocyanate (FITC)labelled goat anti-mouse IgG2a chain specific antibodies (Southern Biotechnology Association, AL) or with a synthetic peptide-fluorescein conjugate for 1 h at 37~ After the final wash cells were resuspended in 1 ml PBS-BSA. Membrane IgG content. Cell suspensions (106 cells/ml) were washed twice with PBS-BSA, and incubated for 40 min at 0~ with FITC labelled goat anti-mouse IgG2a chain specific antibodies (Southern Biotechnology Association, AL) or with a synthetic peptide-fluorescein conjugate. After two washes the cell suspensions were fixed with 0.25% paraformaldehyde (BDH, Dorset, UK). DNA content~cell cycle analysis. Cell suspensions (2.5 • 106 cells/ml) were incubated with BrdU (Sigma, St. Louis, MO) at a final concentration of 10 btM for 1 h at 37~ Next the ceils were washed with PBS, contain'_mg 0.2%. BSA (PBS-

BSA-0.2%) and .incubated in 70%. ethano$ (,I ml) at 0~ After two washes with.PBS-BSA-0.2% the cells 9 incubated for 20 m i n with 1M hydrochloric acid at 0~ The cells were then washed with PBS-BSA-0.2% and incubated w4th. a;nti-Brd,U, monoclonal antibodies (20 .bt.1; Bect0fl. Dickinson BV, EttenLeur, The Netherlands) for 30 rain at 0~ Subsequently, the cel~ls, were washed with PBS-BSA-0.2 % and incubated wifh FITC labelled goat anti-mouse I~-~31 antibodies. (20 BI; Southern Biotechnology Association, Bir~ mingham, AL) for 30 min at 0~ After the final' washes and resuspension in 0.5. ml PBS-BSA: 0.2% the ceils were incubated for 10 rain with propidium iodide (2.6 btl; 1.9 m~ml; Sigma, Sf, Louis, MO). 9

It

r

Results and discussion

Mab production In order to determine the applicability of the FC analysis three suspension batch cultures of the M N I 2 hybridoma cell line were performed in spinner flasks. Since the results of the three batch suspension cultures ~were quantitatively. simi.l;ar, the data of one batch culture are presen~ted and. discussed. The growth;curve and Mab concentration in the culture fluid are shown in Fi:g. ~. It is. seen that exponential growth occurs from day 2..to day 3 of the cel.l culture~ After .the max~mum.eell, density was reac~ed at day. 3 the .number..of non-viable cells (dete~fin. ed with trypan. 9149 exclusion) 9increased.significantl~,~ w~,'.l'e -the-IgG; concentration in the culture:.fluid increased',fi'om~. I0 Ixg/ml to. 28 ~tg/ml..This suggests..that.a~sub-. stantial part of the,IgG is released into, the' culture, fluid by 9149 cells. Velez.et, aL,(l.9.86), alsi~: described the release of Mabs~by,non~fabl~cel,~s;. However~ they stated that .non-.v,iable cel.'l~-had: only a minor contribution,..w, ithout~an.y :s~gni,fican~ ce, to the overall antibody production. Long et al, (1988), reported that a decrease~.,'m eel.l, viabi,l,ity~ was reflected in a reduced 9antibody produ.c[i0n,,. suggesting that. antibody producfion-~is;".as~'oeiat~i~ with the level-,:of:v.iabflity..

68 1500

40

50

A A

1200

40

E

30

E o~

|

v

\

o

%

900

30

"....

E o :G

ii c

20

t.-4"

r-

.o

600

20

E

:3 r = o

o r0 o .D

=E 10

5; 300

10 l

l

I

I

I

I

I

10

20

30

40

50

60

70

0~ z s ~ , & ~ 1

Inllgrml

0 2

3

4

Vilbll

coils

80

(Millions)

(cldllL~

5

40

c u l t i v a t i o n time (d)

B

Fig. I. Growlh curve and Mab concenlration of hybridoma MNI2 in a batch culture. (+) Viable cells ml-I; (z~) non-viable cells ml -I (O) Mab concentration (lag ml-l).

30 E

== In order to determine whether the increased antibody concentration in the culture fluid was due to the release o f tile Mab by non-viable ceils or by actively secreting viable cells, the relation between the integral of viable cells and Mab concentration in the culture fluid was determined according to Renard et al. (1988) (Fig. 2A). The slope o f this line (correlation coefficient r = 0.99) represents the specific secretion rate (4.4 x 10 -7 m g cell -i h-J). The relation between the integral of non-viable cells and Mab concentration in the culture showed no correlation (r = 0.89) (Fig. 2B). The correlation between the integral o f the total cells and Mab concentration was 0.98. This indicates that secretion of Mabs was proportional to the concentration o f the viable cells. Cell size and viability FC analysis was performed to determine cell size and viability, using the forward (FSC) and side

.=_o w ii

20

oc :E 10

o o

i

I

10

20

litll~lrll

flon.vJillb~l

r

(Ill'h/nil)

30

(Millions)

Fig. 2. Correlation between the integral of the viable cell curve (A), non-viable eel/curve (B) and antibody concentration of the MNI2 cell line in a batch culture (r = 0.99).

scatter (SSC) properties of the cells. The FSC is a measure for the relative cell size (arbitrary units) and the SSC is a measure o f cell heterogeneity (arbitrary units). The relative cell size was found

69 to increase during the lag and growth phase of the hybridoma cells and decreased slightly towards the end of the cultivation (Fig. 3). This was in contrast with the results of Frame and Hu (1990). They found an increase of cell size during the lag phase but during the exponential growth phase the cell size remained constant and a decrease was found during the stationary and decline phase. For the estimation of the viability on the basis of scatter properties as measured with FC, two assumptions were made (Sen et al,, 1989): viable cells have a larger relative cell size and are less heterogeneous with respect to cell morphology than non-viable cells, Figure 4 shows the scatter properties o f hybridoma cell line MN12. Population A represents viable cells and population B non-viable cells. Such analysis was performed on samples of the cells at different days of culture. By comparison of the number of cells in the different categories the viability could be determined. While viability was 75% during the first 3

Fig. 3. Growthcurve and relativecell size of hybridomaMNI2

in a batch culture. (§ Viable cells; (O)relative cell size (arbitrary units).

days of culture, it decreased to 18% at d~ty five (Fig. 5). The viability as deterrmned with trypan blue exclusion was 95% during the first 3 days and decreased to 34% after 5 days of culture (Fig. 5). It is seen that FC analysis yields constantly, tower values for the viabitity compared with trypan bltie exclusion. However, the patterns of the viabili~ obtained with the two methods were quite similar. The difference between the FC method and trypan blue exclusion may be due to non-discrimination between small (low FSC) and homogeneous (low SSC) cells. The population is representer by the overlapping area of population A and B (see Fig. 4). This phenomenon somewhat complicated the interpretation of data according to the scatter properties. However, the size measurement method gives a good overall picture of the condition of the hybridoma cells during the culture.

Fig, 4, Dot plot of the scatter properties-of.M~12,~ybridoma cells, relativecell size andcel! h~terbgcn~ityos~measu~d~ith the fl0w cytometer.Popdation ~ cOr/siStof~ialsle"c~ll~;attd populationB :of non~viable:eells;

70

1500

100

1200

80

900

60

E | O

%

0~ >,, I ,.O

.O

E

600

40

300

20

.g

tG) O

0

0 1

2

3

cultivation time

4

5

(d)

the relative cytoplasmic IgG content and the antibody activity showed a discrepancy at day 3. This suggest that at day 3 more heavy chains are synthesized than intact IgG molecules, which bind specifically to the peptide-conjugate. Furthermore, an excess of heavy chains could be toxic for the cells (Ktihler, 1985) and thus affect cell viability at day 4 (see Fig. 5). All the cells contained cytoplasmic IgG and a normal Gaussian distribution was found rather than a bimodal distribution as reported by Altshuler et al. (1986). The mean fluorescence intensity with the antiIgG2a antibody was higher than that observed with the peptide conjugate (data not shown). This difference is probably caused by the different binding properties of the anti-IgG2a-FITC conjugate and the peptide-FITC-conjugate; the polyclonal anti-IgG2a-FITC conjugate may recognize several epitopes on one heavy chain of the IgG molecule, whereas the peptide-FITC conjugate

Fig. 5. Growth curve ((+), viable cells) and viability of hybridoma

1500

100

1200

80

cell line MNI2 as determined with the flow cytometer (V) and by trypan blue exclusion (O). | cO

E Cytoplasmic and m e m b r a n e 1GG content 0

The relative cytoplasmic IgG content and specific cytoplasmic IgG were determined after incubation with fluorescein labelled goat anti-mouse IgG2a antibodies or a peptide-fluorescein conjugate respectively, after permeabilization and fixation with 100% methanol. According to Levitt et al. (1987), fixation with 100% methanol is excellent for preserving both cell morphology and fluorescent staining. Figure 6 shows the results obtained with FITC-conjugated anti-IgG2a antibodies, to determine the relative cytoplasmic IgG content. In the same experiments, the antibody activity of the cytoplasmic IgG was determined using the peptide labelled with FITC. The cytoplasmic IgG content increased during the logarithmic growth phase and decreased rapidly during the decline phase. The two methods agreed to some extent (Fig. 6). Although the ratio between

%

900

60

O~ 0

E .O

E "-z

600

40

O

300

20

~

C | 0

0

1

I

I

I

2

3

4

0

5

cultivation time (d)

Fig. 6. Growth curve ((+), viable cells) and relative cytoplasmic IgG content (fluorescence units) of hybridoma cell lin~ M N I 2 as detected with anti-lgG antibodies (0) and with the peptide-FITCconjugate (4,).

7I binds only to the antigen binding sites at the Fab parts of the IgG molecule. The relative membrane IgG content and relative specific membrane IgG content were also determined, with the same anti-IgG2a-FITC and peptide-F1TC conjugates. The relative membrane IgG content increased during the first day of cultivation,rapidly decreased to day 3, and then remained constant until the end of the cultivation (Fig. 7). A decrease in mean surface fluorescence was also observed by Sen et al. (1990) and Meilhoc et al. (1989). The relationship between mean membrane IgG and specific rate of secretion revealed no correlation (r = 0.03) (data not shown). The relation between cytoplasmic IgG content and specific secretion rate showed also no correlation (r = 0.54) (data not shown). In the literature contradictory results have been reported. Meilhoc et al. (1989) demonstrated also a lack of correlation (r = 0.003) between specific secretion rate and mean surface IgG content. In contrast, Sen et al. (1990) showed a better correlation (r = 0.74).

These observations suggest that a correlation between secretion and surface IgG content may vary between hybridoma cell lines. The decrease in membrane associated IgG during cultivation could be caused at cellular level. The Synthesis of surface IgG and secreted IgG may take place by different pathways. Heavy chains which are secreted and those which are incorporated in the cell membrane are encoded by different rnRNAs (Alt et al., 1980; Ghosh et al., 1987). Thus, a decrease in synthesis rate of surface IgG does not necessarily affect the rate at which IgG is secreted. Another explanation for the decrease of the surface IgG may be the physical shear stress caused by agitation of the cell suspension. Surface IgG which is anchored to the cell membrane by a hydrophobic anchor sequence (Ghosh e t al., 1987), may be stripped from the

Fig. 7. Growth curve ((+), viable cells) and relative membrane

Fig. 8. Dot plot of the determination of the cell cycle distribution of hybridoma cell line MNI2. The red flUorcs~:enci~is a measure for the total DNA content: The green flimmscence is:'a,mc'asum for the S-phase cells.

IgG content (fluorescence units) of hybridoma cell line M N 12 as detected with anti-IgG antibodies (D) and with pcptide-FITCconjugate ( I ) .

72 cell membrane by these shear forces, although this is unlikely at 50 rpm. Cell cycle diso'ibution

The cell cycle distribution was measured to determine the proliferative state of the hybridoma cells. Typical DNA distributions are shown in Fig. 8, and the pattern in time is shown in Fig. 9. The fraction of S-phase cells increased to 55% during exponential growth and decreased rapidly to 25% after the cells had entered the stationary phase. The fraction of G1/G0-phase cells on the other hand decreased during exponential growth and increased rapidly in the stationary phase. The fraction of ceils in the G2/M-phase remained constant. When these results are compared with the antibody concentration during the cultivation (Fig. 1) and the relative cytoplasmic IgG content (Fig. 6), it appears that IgG is mainly synthesized during the S-phase, but secreted into the culture fluid as the cells go through the Gl-phase. From day 1 to 3 the Mab concentration in the culture fluid increases to 10 lx~ml and the cytoplasmic IgG content doubles, while the cells pass through the S-phase of the cell cycle. At day 4 the major part of the ceils pass through the Gl/G0-phase, while the cytoplasmic IgG content decreases rapidly and the Mab concentration in the culture fluid increases from 10 I.t~ml to 28 ~t~ml. Furthermore, a discrimination could be made between G2-phase cells and M-phase cells on the basis of the relative cell size (Fig. 10), because the cell size continuously increases throughout the cell cycle (Baserga, 1985): M-phase cells > G2-phase cells > S-phase cells > G1/G0-phase cells. A comparison between the cell cycle distribution as determined by the BrdU/PI method (Fig. 8) and by the relative cell size/PI method (Fig. 10) is difficult. The different cell cycle phases (G1/G0, S, G2/M) can be determined appropriate using the BrdU/PI method. The advantage of the relative cell size/PI method is the discrimination of G2 and M phase cells according to their different cell size. An appropriate determination of the other cell cycle phases is difficult due to the existence of S-phase cells with a low and a larger cell size.

This is the reason why the number of cells in the G2 and M phase presented in Fig. 10 seems to be higher than in Fig. 8. Although, the increasing trend of the cell size during the cell cycle is clear. The increasing cell size as a function of the cell cycle was also observed by Needham et al. (1991 ). Needham et al. (1991) could distinguish five distinct morpholOgies as a function of the cell cycle. Cells in the early Gl-phase were characterized by their smooth, spherical appearance and small size. Late Gl-phase and S-phase cells were characterized by irregular shapes with small membrane blebs. G2-phase cells appeared to be large rough spheres and the M-phase cells were large smooth cells with a patterned interior. Finally, the characteristic mitotic doublet was observed. Determination of the cell cycle distribution and the proportion of cells in the S-phase provides information on growth dynamics of cultured cells. Besides, cell cycle analysis could give more insights into g o w t h requirements of eukaryotic cells, in terms of nutrients (glucose, glutamine

~..

1500

100

1200

80

900

60

E

"• o

"~

600

40

300

20

0 1

, 2

= 3

cultivation time

I 4

0 5

(d)

Fig. 9. Growth curve and cell cycle distribution oflhybridoma MNI2 during a batch culture. (+) Viable cells; (O) S-phase cells; (&) G I/G0 phase-cells; (O) G2/M-phase cells.

~3 and Dr. W. Jisk0ot for .critically. madi.ng, the manuscript and helpful discussions.

References

FLq. IO. Discrimination of the different cell cycle phases by measurement of relative DNA content and relative cell size.

and other essential amino acids) or serum (trace elements, fatty acids, hormones). Starvation of one of the essential g'rowth requirements can arrest cells in a specific cell cycle phase (Baserga, 1'985). In conclusion, flow cytometry appears to be a useful tool, not only to determine changes (relative cell size, cell cycle distribution) at the cellular level, during the cultivation of hybridoma ceils, but also to determine changes in synthesis of the monoclonal antibodies at single cell level (relative cytoplasmic and membrane IgG content). The IgG seems to be mainly synthesized during the S-phase, but secreted into the culture fluid as the cells go through the G 1-phase.

Acknowledgments The authors wish to thank Dr. G.F.A. Kersten,

Alt FW, Bothwell ALM, Knapp.M,.Siden.E; Mather E;,K0sh~ land M and Baltimore D (.1980)Synthesis of secreted.and membrane bound, immunoglobulin,u hea~j,chains is, directed by mRNAs that differ at their 31 ends...Cell. 20"; 293~-,301i,.. Altshuler GL, Dilwith R, .Sowek J. and Belfort.G (1;986)-Rybridoma analysis at cellular level. Biotech~ Bioeng. Syrup.. :17: 725-736. Aubry J-P, Durand I, De Paoli P and Banchereau J,(.I,990) 7-Amino-4-methylcoumarin-3-aciticacid-eonjugated.;strepta: vidin permits simultaneous .flow cytometry analysis Of either three cell surface antigens or one cell surface .antigen .as a function of RNA and DNA content.. J. Immuno!.=Methods 128: 39-49. Baserga R (1985) The Biology of Cell Reproduction. Harvard University Press, London. Clevenger CV, Bauer KD and Epstein At. C1985):A methofftOr simultaneous nuclear immunofluorescence,and DNA content quantification using monoclonal .antibodies.and, flow cyt0metry. Cytometry 6: 208-214. Dangl JL and Herzenberg LA (1982)Selection of hybfidomas and hybridoma variants using the fluorescence aCliivatedrcell sorter. J. Immunol. Methods 52.: I-.14. Dean PN, Gray JW and Dolbeam PA (.1.982) The.analysis.,and interpretation of DNA distributions, measured by .flow eyto~ metry. Cytometry 3: 1.88-195. Dolbeare F, Gratzner H, Pallavicini MG and Gray JW (!~983) Flow cytometric measurement of total D N A content and incorporated bromodeoxyuridine. Proc. Natl. Acad. Sei;USA 80: 5573-5577. Drijfhout JW, Blnemhoff W, Poolman J,:T and HOogerhotit .:P,. (1990) Solid-phase synthesis and applications:of N-(S,Acetylmercaptoacetyl) peptides. Anal. Bioehem.A:8~- 349,.-354-.: Festin R, Bjorklund~ B, and T0tterman: T (1987~ l~tecti0rr'o~ triple :antibody-binding lyrrlphocytes ~in;standard. Singleflasr flow Cytometry using colloidal, gold, fluorescr pRyco~ erythrin as labels. J.. lmrnunol. Meth0ds !0P .23-~28., Festin R, Bjorklund B and Totterman T (1~990)Single laser: tlbw cytometric detection of lymphocytes binding three antibodies labelled with fluoreseein, phycoerythrin~and a .novel. tandem fluorochrome conjugate. J. Immunol. Methods 126: 69~78. Frame KK and Hu W-S (1990) Cell volume measurement as anestimation of mammalian cell biomass. Biotech..Bi0eng. 36: 191-197. Ghosh SK, Pamaik P and Bankert B (1987) ExpresSii~h"of It"hiii:l' gl membrane forms of immunoglobulin.segregate in. sOmatic cell hybrids. MoL ImmunoL 24: 1335~I=34~3~. Hedley DW and Jorgcnsen HB (1989) Flow cytometric measurement of intraeellular pH i in B I6 tumors, Intercell variance and effect of pretreatment with glucose. Exp. Cell Res. 1~80, 106-116.

74 Hoven MY, De Leij L, Keij JFK and The TH (1989) Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS). J. Immunol. Methods 117: 275-284. Jacobberger JW, Fogleman D and Lehman J (1986) Analysis of intracellular antigens by flow cytometry. Cytometry 7: 356364. KlOppinger M, Fertig G, Fraune E and M iltenburger HG (1991) High cell density perfusion culture of insect cells for production of baculovims and recombinant protein. In: Spier RE, Griffiths JB and Meignier B (eds) Production of biologicals from animal cells in culture. Butterworth-Heinemann Ltd, Oxford, UK, pp. 470---475. Ktihler G (1985) Derivation and diversification of monoclonal antibodies. EMBO J. 4: 1359-1365. Koolwijk P, Rozemuller E, Stad RK, De Lau WBM and Bast BJEG (1988) Enrichment and selection of hybrid hybridomas by pemoll density gradient centrifugation and fluorescentactivated cell sorting. Hybridoma 7:217-225. Kurki P, Ogata K and Tan EM (1988) Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyelin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J. lmmunol. Methods 109: 49-59. Levitt D and King M (1987) Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens. J. Immunol. Methods 96: 233-237. Long WJ, Palombo A, Schofield TL and Emini EA (1988) Effects of culture media on murine hybridomas, definition of optimal conditions for hybridoma viability, cellular proliferation, and antibody production. Hybridoma 7: 69-77. McGuinness B, Barlow AK, Clarke IN, Farley JE, Anilionis A, Poolman JT and Heckels JE (1990) Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria Meningitides; Synthetic peptides define the epitopes responsible for serosubtype specificity. J. Exp. Med. 171; 1871-1882. Meilhoc E, Wittrup KD and Bailey JE (1989) Application of flow cytometric measurement of surface IgG in kinetic analysis of monoclonal antibody synthesis and secretion by murine hybridoma cells. J. Immunol. Methods 121: 167-174. Needham D, Ting-Beall HP and Tran-Son-Tay R (1991) A physical characterization of GAP A3 hybridoma cells: Morphology, geometry and mechanical properties. Biotechnol. Bioeng. 38: 838-852.

Patterson Jr MK (1979) Measurement of growth and viability of cells in culture. Meth. Enzymol. 58: 141-148. Rabinovitch PS, Kubbies M, Chen YC, Schindler D and Hoehn H (1988) BrdU-Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis. Exp. Cell Res. 174: 309-318. Renard JM, Spagnoli R, Mazier C, Salles MF and Mandine E (1988) Evidence that monoclonal antibody production kinetics is related to the integral of the viable cell curve in batch systems. Biotechnol. Lett. 10: 91-96. Rigg KM, Shenton BK, Murray IA, Givan AL, Taylor RMR and Lennard TWJ (1989) A flow cytometric technique for simultaneous analysis of human mononuclear cell surface antigens and DNA. J. Immunol. Methods 123: 177-184. Rink TJ, Tsien RY and Pozzan T (1982) Cytoplasmic pH and free Mg2+ in lymphocytes. J. Cell Biol. 95: 189-196. Sen S, Hu W-S and Srienc F (1990) Flow cytometric study of hybridoma cell culture, correlation between cell surface fluorescence and lgG production rate. Enzyme Microb. Technol. 12: 571-576. Sen S, Srienc F and Hu W-S (1989) Distinct volume distribution of viable and non-viable hybridoma cells: A flow cytometric study. Cytotechnology 2: 85-94. Terstappen LWMM, Meiners H and Loken MR (1989) A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations. J. Immunol. Methods 123:103-112. Velez D, Reuveny S, Miller L and Macmillan JD (1986) Kinetics of monoclonal antibody production in low serum growth medium. J. Immunol. Methods 86: 45-52. Wing MG, Montgomery AMP, Songsivilai S and Watson JV (1990) An improved method for the detection of cell surface antigens in samples of low viability using flow cytometry. J. Immunol. Methods 126: 21-27.

Address for correspondence: J.M. Coco-Martin, Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures.

Flow cytometric (FC) analysis was applied to determine changes at cellular level during the cultivation of hybridoma cell line MN12 in a suspension ba...
873KB Sizes 0 Downloads 0 Views