Showing papers on "Lysis published in 1993"

Preparation of yeast RNA

Abstract: This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample-to-sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.

Rapid DNA extraction protocol from soil for polymerase chain reaction‐mediated amplification

Abstract: A simple and rapid method of DNA extraction from soil was developed and DNA was made suitable for subsequent efficient amplification by the polymerase chain reaction (PCR). Key features of the extraction and purification were cold lysozyme- and SDS-assisted lysis with either freezing-thawing or bead beating, cold phenol extraction of the resulting soil suspension, CsCl and KAc precipitation and, finally, spermine-HCl or glass milk purification of DNA. Crude DNA preparations contained 4–20 μg DNA per g of soil extracted, and at least 50% of this was recovered in the final purified DNA preparations. The resulting DNA was pure enough to be restricted by various enzymes, and was amplifiable at concentrations of up to 20 ng of soil-derived DNA per 50 μl reaction mix. Amplification of a 683 bp target sequence, pat, was performed with different Taq DNA polymerases. Application of the protocol enabled us to detect target DNA derived from roughly 103 introduced Pseudomonas fluorescens (RP4 :: pat) cfu per g of soil. The fate of an introduced population in the soil could be followed to this limit with PCR-assisted detection of target DNA. In addition, target DNA was detected in soil 5 months after release, when the introduced organism was no longer detectable on selective agar plates. The extraction and purification protocol applied to various different soil types resulted in DNA of sufficient purity to permit amplification by PCR.

Gel purification of red algal genomic DNA: an inexpensive and rapid method for the isolation of polymerase chain reaction-friendly DNA

Abstract: A simplified approach for the extraction of DNA from red algae in presented. Procedures are simple and fast, requiring a minimum of reagents and apparatus. The method involves an initial lysis step followed by an optional phenol/chloroform extraction. The final gel-purification step removes polymerase chain reaction-inhibiting polysaccharides from the DNA preparation. DNA is extracted as easily from dried algae as it is from snap-frozen, fresh material, thus greatly facilitating the collection and transport of algal samples.

Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis.

Abstract: We demonstrate here that CD59, an inhibitor of the membrane attack complex (MAC) of the complement system, is present in cell-free seminal plasma (SP) at a concentration of at least 20 micrograms/ml. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and Edman degradation indicated that this protein, SP CD59, was similar, if not identical, to CD59 isolated from erythrocyte (E) membranes (E CD59). Like purified E CD59, SP CD59 also possesses a glycosyl phosphatidyl inositol (GPI) anchor and incorporates into the membranes of heterologous cells where it inhibits lysis by the human MAC. This phenomenon could be demonstrated not only if cells were incubated with purified SP CD59 but also if unfractionated SP were used. Further, CD59 in unfractionated SP bound to washed spermatozoa, increasing their membrane content of the protein. The mechanism by which this protein retains its GPI anchor while apparently present in the fluid phase is of interest and was further investigated. Using the techniques of high-speed centrifugation, fast performance liquid chromatography fractionation, and electron microscopy, we found that all detectable SP CD59 was associated with vesicular extracellular organelles. These organelles, named "prostasomes," were previously known to be present in SP and to interact with spermatozoa, although their function was uncertain. Interaction of heterologous E with prostasomes rendered the cells more resistant to lysis by human MACs. We propose that these organelles represent a pool of CD59 from which protein lost from spermatozoa, perhaps as a result of low level complement attack or of normal membrane turnover, can be replenished.

Islet Cell DNA Is a Target of Inflammatory Attack by Nitric Oxide

Abstract: NO has been identified recently as the prime islet-toxic product of inflammatory macrophages. The adverse effects of IL-1 on isolated islets also have been reported to involve NO. We now show that exposure of an islet cell suspension to the NO donor nitroprusside or to activated macrophages leads to DNA strand breaks. Macrophages did not induce DNA damage in the presence of the NO synthase inhibitor NG-methyl-L-arginine. DNA strand breaks were demonstrated at the level of single cells by a modified nick-translation procedure and confirmed by analysis of DNA fragmentation by gel electrophoresis. DNA strand breaks occurred within 1 h and preceded islet cell lysis. DNA damage could not be prevented by inhibitors of endogenous endonucleases. We conclude that islet cell DNA is an early target of NO action.

Preparation of genomic DNA from mammalian tissue

Abstract: There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteinization and recovery of DNA. The main differences between various approaches lie in the extent of deproteinization and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.

Surface action of gentamicin on Pseudomonas aeruginosa.

Abstract: The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from surface perturbation; the two effects in concert make aminoglycoside drugs particularly effective antibiotics. Images

Cell Wall Structure, Synthesis, and Turnover

Abstract: Walls of gram-positive bacteria are dynamically variable and flexible structures that enclose and protect the underlying cytoplasmic membranes. The wall serves to protect the underlying protoplast, resist turgor, and maintain the shape of the cell. The wall in B. subtilis, like the walls in many other gram-positive bacteria, is composed mainly of peptidoglycan and one or more anionic polymers. These components are synthesized on identical anchor lipids, covalently attached to each other before or during insertion into the wall, processed through the wall, and finally released by turnover while still attached to each other. It is now clear that both types of polymer are essential for normal wall function and morphogenesis. In addition to containing peptidoglycan and anionic polymers, walls of gram-positive bacteria may contain substantial proportions of protein, held either covalently or noncovalently within the peptidoglycan-anionic-polymer complex, together with neutral polysaccharides, lipoteichoic acid, and the cations that form part of the polyelectrolyte gel structure of the wall complex. Teichoic acids found in Bacillus species other than B. subtilis are listed in this chapter. The end of exponential growth in batch cultures of B. subtilis may be followed by marked lysis of the culture. This is due to the action of autolysins, enzymes that hydrolyze either the glycan or the peptide moieties of peptidoglycan. The final stage in the incorporation of peptidoglycan or peptidoglycan-anionic-polymer complex is accomplished by transpeptidation reactions.

Rapid purification of high-activity Taq DNA polymerase

Abstract: The method described here is derived from that of Engelke et al. (1), and uses the same cloned form of Thermus aquaticus {Taq) DNA polymerase to produce this enzyme in E.coli. The modified purification method described here is quite simple, however it is important to note that factors such as the bacterial strain used, induction time and protein concentration during isolation have been optimized. Deviation from the established parameters can have large effects on enzyme yield and activity. E.coli strain INVlalphaF' (Invitrogen Corp., San Diego, California) was transformed with the pTaq plasmid, which contains the Taq gene expressed under control of the tac promoter (1). Large-scale cultures of E.coli containing the pTaq plasmid were initiated by adding 500 n\ of an overnight culture to one litre of LB broth with ampicillin (80 mg/1). These cultures were grown at 37°C for 11 hours to an ODeoo of approx. 0.8, and then IPTG was added to a concentration of 125 mg/1. After 12 hours of induction the cells were harvested by centrifugation and washed in 100 ml per litre of original culture volume of buffer A (50 mM Tris-HCl pH 7.9, 50 mM dextrose, 1 mM EDTA; all reagents used in the purification were molecular biology grade, and care was taken to avoid contamination with biological material or metal ions). Cells were recovered by centrifugation and resuspended in 50 ml per litre of original culture volume of pre-lysis buffer (buffer A plus 4 mg/ml lysozyme). After 15 minutes at room temperature an equal volume of lysis buffer was added (10 mM Tris-HCl pH 7.9, 50 mM KC1, 1 mM EDTA, 1 mM PMSF, 0.5 % Tween 20, 0.5 % Nonidet P40) and the lysis mixture was incubated in 200 ml aliquots in pyrex flasks at 75 °C for 1 hour. The lysis mixture was then transferred to plastic bottles for centrifugation at 15,000 rpm for 10 minutes at 4°C, and the clarified lysate was transferred to a clean pyrex flask.

Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant.

Abstract: To probe the potential for extracellular degradation of glycoprotein oligosaccharides in conjunction with Chinese hamster ovary (CHO) cell culture, an initial characterization of several CHO cell glycosidases was performed using 4-methylumbelliferyl substrates. CHO cell lysates contained sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities with pH optimums near 5.5, 4, 6, and 6.5, respectively. These glycosidase activities were also present in cell-free supernatant samples from commercial CHO cell cultures. The sialidase activity was further characterized. In contrast to previous reports concerning mammalian sialidases, the sialidase activity in CHO cell lysate retained considerable activity at pH 7 and was very stable, with a half-life of 57 h at 37 degrees C. Both the Km and Vmax of CHO lysate sialidase for 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) varied with pH, and this activity was competitively inhibited by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid and by free N-acetylneuraminic acid. The kinetic characteristics and pH-activity profiles of the CHO cell lysate and cell culture supernatant sialidase activities were essentially identical, and both released sialic acid from the glycoprotein fetuin at pH 7.5. These results suggest that the oligosaccharides of glycoproteins secreted by CHO cells can potentially be modified extracellularly by sialidase under culture conditions which promote the release and extracellular accumulation of this enzyme.

Anaerobic waste-activated sludge digestion - A bioconversion mechanism and kinetic model

Abstract: The anaerobic bioconversion of raw and mechanically lysed waste-activated sludge was kinetically investigated. The hydrolysis of the biopolymers, such as protein, which leaked out from the biological sludge with ultrasonic lysis, was a first-order reaction in anaerobic digestion and the rate constant was much higher that the decay rate constant of the raw waste activated sludge. An anaerobic digestion model that is capable of evaluating the effect of the mechanical sludge lysis on digestive performance was developed. The present model includes four major biological processes-the release of intracellular matter with sludge lysis; hydrolysis of biopolymers to volatile acids; the degradation of various volatile acids to acetate; and the conversion of acetate and hydrogen to methane. Each process was assumed to follow first order kinetics. The model suggested that when the lysed waste-activated sludge was fed, the overall digestive performance remarkably increased in the two-phase system consisting of an acid forming process and a methanogenic process, which ensured the symbiotic growth of acetogenic and methanogenic bacteria.

Inhibition of contractile vacuole function in vivo by antibodies against myosin-I

Abstract: MYOSIN-I is thought to supply the force for movement of cell membranes relative to actin filaments (reviewed in refs 1, 2), but confirmation of this hypothesis has been difficult because of the presence of multiple isoforms of myosin-I and other unconventional myosins in most cells3. We report here the first evidence that a myosin-I isoform is essential for a specific class of intracellular membrane movements in vivo. In Acanthamoeba, the contractile vacuole is an autonomous structure which fuses with the plasma membrane to control the water content of the cell. Because myosin-IC is the only myosin-I isoform concentrated in the contractile vacuole complex4,5, and a protein antigenically related to myosin-IC is located on or near the Dictyostelium (slime mould) contractile vacuole6, we thought this organelle might provide the best opportunity to demonstrate a relationship between myosin-I and membrane motility. Antibodies that inhibit the activity of Acanthamoeba myosin-IC in vitro interfere with expulsion of excess water by the contractile vacuole in vivo, leading to overfilling of this organelle and cell lysis. Myosin-IC may generate the force required to contract the vacuole and may also be involved in transfer of water to the contractile vacuole during refilling.

Role of TNF-alpha in CD8+ cytotoxic T lymphocyte-mediated lysis.

Abstract: The possibility that lymphokines such as TNF-alpha produced by CD8+ CTL are responsible for acute (short term) target cell damage induced by CTL has been debated for many years. However, the slow kinetics of TNF-induced target cell death stands in sharp contrast to the rapid target cell lysis mediated by CTL. We find that cloned CD8+ CTL activated through their TCR secrete TNF-alpha. On the other hand, our cloned CTL also have a membrane form of TNF-alpha, and they kill TNF-alpha-sensitive target cells not recognized through the TCR in a slow (18-h) lytic reaction using this surface-associated TNF-alpha. There is no secreted TNF-alpha release during this interaction. Cyclosporin A and protein synthesis inhibitors block TNF-alpha secretion, but have no effect on slow lysis mediated by the CTL. On the other hand, TNF-alpha-resistant variants are greatly resistant to slow lysis, and antibodies to TNF-alpha strongly inhibit this slow lysis. Thus, although secreted TNF-alpha does not seem to be the mechanism behind slow lysis, some form of TNF-alpha, most likely the membrane-associated form, must be involved. Not only does surface TNF-alpha appear to be biologically active in these CTL, but its expression is enhanced severalfold upon activation of the CTL through the TCR. This may be important in vivo, where surface TNF-alpha could preserve the localized nature of cytolysis and endow a CTL with an additional, albeit slower, mechanism of cell lysis. Finally, we find that although activated CTL clearly use the membrane form of TNF-alpha in slow lysis, they appear not to use TNF-alpha, in any form, during acute lysis, even under conditions in which degranulation and perforin assembly are blocked.

The effect of phagocytosis of poly(L-lactic acid) fragments on cellular morphology and viability

Abstract: The aim of this study was to investigate the effect of phagocytosed poly(L-lactic acid) particles on the morpholgy and viability of phagocytes, mainly macrophages. Therefore, predegraded poly(L-lactic acid) (P-PLLA) and nontreated PLLA (N-PLLA) particles, both having diameters not exceeding 38 µm, were injected intraperitoneally in mice. P-PLLA particles were obtained by 25 kGy γ-irradiation of N-PLLA particles. N-PLLA and P-PLLA particles were injected using an 0.3% ethanol/0.9% saline solution intraperitoneally to the mice. We also studied the release of the absorbed ethanol as a possible model for the release of low molecular weight, potentially toxic products. As control, nondegradable polytetrafluoroethylene (PTFE) particles and the carrier solution were used. After 1, 2, 3, 4, 5, and 7 days, the cells of the abdominal cavity were harvested to study the effect of phagocytosis of polymer particles on phagocytic cell morphology and viability. Studies with transmission electron microscopy indicated that, upon injection of particles in the peritoneal cavity, macrophages demonstrated signs of cell damage, cell death, and cell lysis due to phagocytosis of a large amount of P-PLLA particles. The morphology of the cells that had phagocytosed the N-PLLA and PTFE particles did not differ substantially from those of control animals in which only the solution was injected. Also, in the controls, hardly any cell death and no debris was observed. When the PLLA particles were injected as a suspension in a 0.3% ethanol/0.9% saline solution, no difference was observed between N-PLLA and P-PLLA. After phagocytosis, both cause cell damage, sometimes leading to cell death. The highest numbers of necrotic cells were observed on day 2. The effects could be caused by the (peak) release of degradation products from P-PLLA fragments or by the release of the absorbed ethanol when the 0.3 ethanol/0.9 saline solution was used to administer the particles. In conclusion, it can be stated that cell damage, sometimes leading to cell death, may be caused by phagocytosed poly(L-lactic acid) particles.

Lectin-induced apoptosis of tumour cells

Abstract: The mechanisms of cytotoxic activity of Griffonia simplicifolia 1-B4 (GS1B4) and wheat germ agglutinin (WGA) lectins against various murine tumour cell lines were studied. Tumour cells that lack lectin-binding carbohydrates were resistant to lysis by these lectins. However, YAC-1 cells that expressed GS1B4 lectin-binding sites showed low sensitivity to lysis. To further analyse the relative importance of cell surface carbohydrates in lectin cytotoxicity, BL6-8 melanoma cells, which do not express the alpha 1,3 galactosyltransferase (alpha 1,3GT) gene and cell surface alpha-galactosyl epitopes reacting with GS1B4 lectin, were transfected with cDNA encoding alpha 1,3GT. After transfection, BL6-8 cells expressed high levels of GS1B4-binding alpha-galactosyl epitopes, but remained resistant to lysis by GS1B4 lectin, suggesting that the presence of lectin-binding epitopes, while essential, is not sufficient for tumour cell lysis and probably some intracellular mechanisms are involved in the regulation of lectin-mediated cytotoxicity. We found that the GS1B4 and WGA lectins induced apoptosis with DNA fragmentation of sensitive, but not resistant, tumour cell lines. DNA fragmentation, as well as tumour cell lysis, was blocked in the presence of the specific inhibitory sugar. To determine whether binding of the lectin to cell surface carbohydrates is sufficient to trigger tumour cell lysis, lectin-sensitive CL8-1 melanoma cells were incubated with GS1B4 lectin immobilized on agarose beads. Although these tumour cells bind to the immobilized lectin, it failed to trigger tumour cell death, suggesting that only soluble lectin is capable of tumour cell lysis and lectin internalization is probably required for their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition.

Abstract: We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition. Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content. Phospholipids composed 0.5-13% of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature beta-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

Macrophage-generated nitric oxide as cytotoxic factor in destruction of alginate-encapsulated islets. Protection by arginine analogs and/or coencapsulated erythrocytes.

Abstract: Isolated rat islets were microencapsulated in alginate beads of about 1.5 mm in diameter. These were cocultured with activated or resident peritoneal macrophages of syngeneic rats for 24 hr. Examination of the encapsulated islets by transmission electron microscopy showed that the islets were lysed by activated (80.0 +/- 12.8% of islets lysed), but not by resident, macrophages (17.5 +/- 12.2% lysis) despite encapsulation. Islet lysis was inhibited in a concentration-dependent manner by a specific nitric oxide-synthase inhibitor (0.5 mM NG-methyl-L-arginine: 5.9 +/- 3.9% lysis) in an L-arginine-reversible manner (0.5 mM NG-methyl-L-arginine + 10 mM L-arginine: 55.1 +/- 16.6% lysis). Incubation of encapsulated islets with 3 different nitric oxide-generating compounds also resulted in a concentration-dependent islet lysis. Coencapsulation of autologous erythrocytes was found to be an effective and easy way of protection from macrophage-mediated lysis. Protection was dependent upon the number of erythrocytes coencapsulated. This in vitro study demonstrates that nitric oxide secreted by activated macrophages is able to destroy islets despite encapsulation in alginate, and that both, inhibition of nitric oxide formation using enzyme inhibitors and scavenging of nitric oxide once formed exploiting the hemoglobin of autologous erythrocytes, protect encapsulated islets from destruction.

A Mechanism for Neutrophil-mediated Lysis of Human Neuroblastoma Cells

Abstract: Neutrophils mediate the lysis of human neuroblastoma cells coated with human/mouse chimeric anti-GD2 ganglioside antibody ch14.18. This study examined the mechanism(s) by which this occurs. Neutrophil degranulation was found to be a required step for lysis, since release of granular enzymes from neutrophils correlated with the lysis of antibody-coated neuroblastoma cells. In addition, agents which block degranulation specifically inhibited this process. Antibody-dependent lysis of neuroblastoma cells was enhanced by exposing neutrophils to granulocyte-macrophage colony stimulatory factor. An increased release of lytic granular molecules was found to be responsible for this lymphokine-mediated phenomenon. Among the molecules released from neutrophil granules that were shown to be involved in neuroblastoma cell lysis were defensins, M(r) 3000-4000 neutrophil granular proteins which are known to bind and permeabilize tumor cells. In addition, cathepsin-G, a neutrophil granular protease, was demonstrated for the first time to mediate the lysis of human neuroblastoma cells. The enzymatic activity of cathepsin-G was found to be required for the lysis of these tumor cells, since phenylmethylsulfonyl fluoride blocks the lytic ability of this protein.

The missing link in phage lysis of gram-positive bacteria: gene 14 of Bacillus subtilis phage phi 29 encodes the functional homolog of lambda S protein.

Abstract: In most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane. The lesion is brought about by a second phage-encoded lysis function. For the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium. Thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive. Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli. Production of protein 14 in E. coli resulted in cell death, whereas production of protein 14 concomitantly with the phi 29 lysozyme or unrelated murein-degrading enzymes led to lysis, suggesting that membrane-bound protein 14 induces a nonspecific lesion in the cytoplasmic membrane.

Methods and compositions for isolating nucleic acids

Abstract: Compositions and methods for isolating nucleic acids from biological tissues and cells and for tissue/cell solubilization for other molecular biological uses, wherein the compositions comprise, in part, novel combinations of chaotropic agents and aromatic alcohols which act synergistically to effect better tissue/protein solubilization. The inventive compositions further include aprotic solvents for deactivation of ribonucleases and denaturization of proteins, as well as detergents for enhancing cell lysis and nucleoprotein dissociation. The inventive methods also comprise the use of a centrifuge, a solid-support matrix, and a microporous membrane for final isolation of the precipitated nucleic acids, resulting in high yield and purity of the precipitated nucleic acid.

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

Abstract: The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h−1 to 0.01 h−1 when decreasing the pH from 7 to 6.8.