Capillary zone electrophoresis of organic acids in serum of critically ill children.

Capillary zone electrophoresis with indirect UV detection was found to be suitable for the determination of organic acids in serum. Serum can be analysed directly without any deproteination in a capillary coated with linear polyacrylamide. With 10 mM epsilon-aminocaproic acid-10 mM mandelic acid (pH 3.8) as the operational electrolyte, anions such as pyruvate, phosphate, citrate, malate, acetoacetate and lactate can be determined in 12 min. In quantitative analysis, the calibration line for lactate is linear over the range 0-10 mM. The detection limit for citrate was 8 microM. The effect of the chloride concentration on the migration times of minor peaks is discussed. The potential of the method was demonstrated by analysing sera from several critically ill children.

Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures.

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical "ladder" of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.

Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: effect of nutrient concentration.

The iron-rich (500 microM ferric citrate) protein-free supplement was added to six different basal media. Cell growth and monoclonal antibody production of a mouse-mouse hybridoma were investigated in 1.3 1 batch cultures performed in a laboratory bioreactor with automatic control of pH and dissolved oxygen concentration. RPMI 1640 served as the control medium. Fortification of the basal medium by balanced mixtures of amino acids and vitamins showed higher positive effect than daily supplementation by glucose and glutamine. Strongly fortified medium, based on RPMI 1640, was found superior to other basal media. The viability index increased by a factor of 3.04 and the total antibody production by a factor of 2.82, relative to the control.

Nucleosomes occurring in protein-free hybridoma cell culture. Evidence for programmed cell death.

In addition to monoclonal immunoglobulin, two kinds of nucleoproteins, NP1 and NP2, were isolated from the supernatants of hybridoma cultures set up in a protein-free medium. As shown by SDS-electrophoresis the two nucleoproteins shared a set of proteins (apparent Mr 11,000 to 15,000), and differed in the DNA moiety (approximately 150 bp in NP1, approximately 350 bp in NP2). The amino acid composition of the protein moiety confirmed the nucleosomal origin of NP1 and NP2. The findings support the view that in hybridoma cultures the cells undergo death by apoptosis, i.e. a programmed process characterized by initial fragmentation of chromatin.