Batches Of Serum Research Articles

Effects of serum batches on the incidence of chromosomal abnormalities

Effects of serum batches on the incidence of chromosomal abnormalities

Factors influencing the response of MCF-7 cells to an agonist of luteinising hormone-releasing hormone

To clarify the mechanism by which the luteinising hormone-releasing hormone agonist, buserelin, may have direct effects on breast cancer cells, factors potentially influencing its action have been studied in the MCF-7 breast cancer cell line. Oestradiol and epidermal growth factor (EGF), which stimulate the growth of MCF-7 cells in culture, reversed, at least in part, the inhibitory effects of buserelin. Insulin also abolished growth inhibition. Quantitative effects of buserelin differed according to the batch of fetal calf serum used as media supplement. These data suggest that the direct inhibitory effects of buserelin on breast cancer cells are mediated at least in part by an antagonism of growth-promoting factors.

In vitro cultivation of adult Litomosoides carinii: evaluation of basic culture media, gas phases and supplements

Adult Litomosoides carinii, recovered from cotton rats (Sigmodon hispidus) 4-5 months post-infection (p.i.), were cultivated in vitro with emphasis on investigations into the development of intra-uterine embryonic stages. Baseline values for the embryonic status of female worms were established immediately after recovery from the hosts. In such females, on average, 16% of the intra-uterine stages were fully formed microfilariae while the remainder belonged to the early embryonic classes that were characterized. For the evaluation of culture success, apart from survival of worms in vitro, the rate of microfilariae development (mf rate) served as a major parameter. Of the five basic culture media RPMI 1640, F12, L15, NCTC 135, and IMDM, tested without supplementation, RPMI 1640 yielded by far the best results (survival = 14 days; mf rate = 41%), and was therefore chosen as the routine medium. In comparison with 5% CO2 in nitrogen, a gas phase of 5% CO2 in air was superior, although the resulting oxygen tension of 138 mmHg in the medium was not physiological. Addition of 10% newborn or foetal bovine serum to the medium in some cases distinctly influenced results. Effects of different batches of sera varied from 'filaricidal' to 'very promoting'. Co-cultivation of worms and Sigmodon cells, or rhesus monkey LLCMK2 cells, only marginally improved results. Of the serum substitutes Ultroser G, BMS, and Clex, the latter had a moderately promoting effect. The protein supplements bovine serum albumin, transferrin and haemoglobin significantly improved results, and could replace certain batches of serum. Supplementation with the haemin moiety alone was less effective than with haemoglobin. The anti-oxidants glutathione plus ascorbic acid proved beneficial in combination with a serum supplement only. Results from other experiments with multiple supplementation also suggest that various supplements may act only in a synergistic manner. The longest average time that adult L. carinii survived in vitro was 3-4 weeks. The highest mf rate was 78%, which indicated that all normal embryonic stages present in the uteri of a female at the start of culture completed their development to microfilariae, however, oogenesis ceased in vitro. The parameters for embryonic development employed proved to be highly sensitive for the judgment of various culture conditions.

The influence of contamination of culture medium with hepatitis B virus on the outcome of in vitro fertilization pregnancies

Heat-inactivated human serum is added to the culture medium used for in vitro fertilization and other forms of assisted conception. Because one batch of pooled serum contained hepatitis B virus, an epidemic occurred among women participating in the treatment program. Seventy-nine women had serologic proof of hepatitis B infection. This incident gave the opportunity to study the effect of hepatitis B virus on pregnancy outcome and the newborn. The situation is unique because the preimplantation embryo was exposed to hepatitis B virus or the pregnancy was complicated by a (sub)clinical infection. Twenty-four women were or became pregnant while having an acute hepatitis B infection. Five pregnancies ended in abortion. The remaining 19 pregnancies ended in the birth of 24 children. No evidence for any harmful effect of exposure to hepatitis B virus in the embryonic or fetal period on the newborn could be found.

Interlaboratory evaluation of three culture media for postimplantation rodent embryos

The first aim of the study was to compare the ability of rat serum, human serum, and a mixture of human and rat serum (4:1) to support in vitro development of rodent postimplantation embryos. The comparison was made in three laboratories using rat embryos and in one laboratory using mouse embryos. Batches of sera, initial developmental stage, duration of culture, and endpoints were identical in the laboratories. The second aim of the study was to evaluate if other variables that could not be standardized would significantly influence the results of the laboratories. No reproducible difference was observed among the culture media or among the laboratories except that growth and differentiation were slower in the laboratory using mouse embryos. Further experiments are needed to exclude small differences in performance of the media.

Colony promoting activity: differences from interleukin-3 and similarities to interleukin-6

The supernatant of long-term bone marrow culture contains colony promoting activity (CPA) which does not have granulocyte-macrophage colony stimulating activity (CSF) but which enhances granulocyte-macrophage (CM) colony formation in the presence of CSR CPA might consist of IL-3 or IL-3-like activity, since CPA stimulates proliferation and differentiation of more immature cells to CSF-responding granulocytes-macrophage progenitors (GM-CFC), and since IL-3 also stimulates immature hemopoietic cells to proliferate and differentiate to functional blood cells. IL-1 and IL-6 are also known to enhance GM colony formation. One or both of these molecules can accordingly be another candidate for CPA. In the present study, GM-CSF activity of IL-3 was dependent on the batch of serum: it was negative in the presence of fetal calf serum, but positive in the presence of horse serum. In contrast, GM-CSF and CPA showed no such dependence on the batch of serum. The addition of IL-3 to GM colony assay system did not enhance but rather suppressed GM colony formation. The supernatant of long-term bone marrow culture which contains substantial levels of CPA did not stimulate proliferation of IL-3 dependent DA-1 cells, but facilitated the proliferation of IL-6 dependent, MH60-BSF2 cells. No detectable level of IL-1 activity was found in the supernatant. These results indicate that CPA is different from IL-1, IL-3 or GM-CSF, but similar to or the same as IL-6.

Purification of C8 and C9 from Rat Serum

A procedure based on modifications of published methods for human proteins for the isolation of rat C8 and C9 from one batch of serum is described. The procedure allows the rapid, large-scale isolation of pure and haemolytically active proteins. Rat C9 had a slightly higher molecular weight than human C9 on SDS-PAGE and similar isoelectric point. Rat C8 differed from human C8 in the molecular weight of the gamma chain (23,000 and 21,000 kD respectively), and on isoelectric focusing (pI rat C8: 6.5-6.9; pI human C8: 7.4-7.9).

Discrimination of fetal bovine serum from other sera by silver staining or lectin blotting of SDS/PAGE-separated serum proteins.

In order to develop a methodology to discriminate fetal bovine serum from other sera with which it might be contaminated, proteins from fetal bovine, newborn calf, colostrum-free newborn, and calf sera were separated by SDS/PAGE and analyzed by silver staining of the gels or lectin blotting, using concanavalin A (Con A), following transfer of the proteins to nitrocellulose. A high molecular weight Con A-reactive glycoprotein of an apparent molecular mass greater than 200,000 daltons was present in newborn, colostrum-free, and calf sera, but absent or at a significantly lower concentration in fetal serum. These two methods accurately and reliably identified contamination of fetal serum with other sera in a series of blind studies on unknown, coded samples. As little as 1% colostrum-free serum in a batch of fetal serum can be detected by either procedure.

Short and long term stability of the elemental composition of human body fluid reference materials and their use as master lots

Studies have been made of both short and long term stability of trace elements in lyophilized human body fluid reference materials, as well as the stability of mercury in reconstituted urine solutions. No detectable concentration changes for mercury, lead and aluminium occurred during the 5-year period. There are large differences in the amounts of mercury loss among different reconstituted materials. Addition of traces of gold to the solutions minimized the mercury loss and increased the useable time of the reconstituted material from hours to 8 days. A “certification process” based on direct determinations by reference laboratories and by comparison against master lots of the same material and against similar certified reference materials is presented. Values for calcium, copper and mercury obtained from reference laboratories and using the data transfer principle have been assigned in new batches of urine and serum.

Detection of influenza a type‐specific antibodies in chicken and turkey sera by enzyme linked immunosorbent assay

An ELISA test for detection of influenza A virus type-specific antibodies in chicken and turkey sera is described. Antigen was prepared from infected allantoic fluids using an H7N1 virus. Virus particles were disrupted using sodium deoxycholate to release internal antigens and tests were standardised by testing batches of sera from influenza-free birds. A negative antigen was included in the test and was shown to be effective in eliminating the non-specific increase in absorbance values observed with influenza-negative sera from older birds. Sensitivity and group-specificity were demonstrated by infection of chickens and turkeys with influenza subtypes unrelated to the H7N1 virus from which the antigen was prepared, so that only type A specific responses were being detected. The sensitivity was equivalent to the haemagglutination-inhibition test. Antisera to all 13 haemagglutinin subtypes, prepared in chickens, were strongly positive, again highlighting the group reactivity of the test and demonstrating its suitability as a screening test.

Spontaneous release of acetylcholine affects the physiological nicotinic responses of rat retinal ganglion cells in culture

Nictonic cholinergic responses have been previously documented in mammalian retinal ganglion cells. In the present study, dissociated retinal cells were densely plated and grown in a specific batch of rat serum. When held at negative potentials during whole-cell recording with a patch electrode, the retinal ganglion cells located close to presumptive cholinergic amacrine cells in these cultures were found to respond to acetylcholine (ACh; 20-200 microM) with an apparent outward current in the presence of physiological salines on both sides of the membrane. Other nicotinic agonists (nicotine, carbachol) produced the same effect. Conductance measurements revealed that this apparent outward current was actually a decrease in a tonic inward current. Nicotinic antagonists such as d-turbocurarine (10 microM) and dihydro-beta-erythroidine (20 microM), when applied in dishes that had never been exposed to exogenous ACh, produced a similar decrease in a tonic inward current. The reversal potential of the tonic current suppressed by ACh was similar to the nicotinic current previously studied in these central neurons. Furthermore, purified acetylcholinesterase was capable of modulating the tonic inward current of the retinal ganglion cells. Lowering an excised patch of muscle into dense areas of the retinal cultures activated nicotinic channels in the muscle membrane, indicating the presence of endogenous ACh in the culture fluid. Divalent cations such as Co2+ blocked the tonic inward current of retinal ganglion cells in these cultures. Finally, direct biochemical measurements indicated that low levels of endogenous ACh (on the order of 0.5 microM near putative cholinergic amacrine cells) were present in the retinal cultures. Taken together, these results show that ACh was being spontaneously released into these cultures, resembling at least to some degree the tonic leakage of ACh found in the intact retina. This concentration of ACh was capable of tonically depolarizing the membrane potential of retinal ganglion cells exposed to this dosage. This culture system allows the study of trophic effects of ACh on a central mammalian neuron in a precisely controlled extracellular environment.

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.

A cell attachment assay for use in the standardization of serum products

A culture tube assay has been developed which can be used to measure relative amounts of cell attachment activity present in different batches of serum or serum products. The assay utilizes a transformed line of BHK-21 cells which is highly dependent upon serum factors for attachment when the cells are subjected to mild liquid shear forces (tube rotation of 1 rpm). Approximately fivefold differences in attachment activity were observed between different batches of bovine calf serum, while up to 20-fold differences were observed between different batches of horse serum. Less than twofold differences were seen between different lots of fetal bovine serum. The assay appears to have application as a quality control measure for screening serum products.

Epidermal growth factor enhances N-ethyl-N-nitrosourea-induced morphological transformation of Syrian hamster embryo cells.

To improve the usefulness of the Syrian hamster embryo (SHE) cell transformation system as a short-term test, it was investigated whether the variation in results due to serum variability could be reduced by the addition of epidermal growth factor (EGF). It was found that EGF-significantly (3-7-fold) enhanced the frequency of morphological transformation induced by N-ethyl-N-nitrosourea or benzo[a]pyrene if added to growth medium supplemented with a batch of serum which had a low ability to support transformation. Furthermore, addition of EGF to the assay medium enabled the demonstration of dose dependence of transformation with relatively small group sizes (up to 2000 colonies). Finally, it was observed that the transformed phenotype was easier to recognize in the presence of EGF. These data suggest that routine addition of EGF to the assay medium might reduce variability and enhance sensitivity of the SHE transformation assay.

Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation

The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300–1000 nM. Growth-retarding effects of selenite became apparent at 300–2000 nM, showing a large cell line variation. Supplementation with 50–100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.

A serum-free medium for culturing lactogen dependent and autonomous NB2 node lymphoma cells.

A serum-free, hormone-free medium (SF2) was designed for the Nb2 rat lymphoma bioassay for lactogens as batches of horse serum (HS), which were commonly used, were found to be inconsistent in their suitability and to contain factors modulating the PRL-induced growth response of clone Nb2-11C. In a 3-day incubation with less than 500 pg/ml human GH (hGH), SF2 was better than the traditional medium in supporting Nb2-11C growth, although the comparative efficiency of SF2 decreased at higher hGH levels. Known growth factors (epidermal growth factor, fibroblast growth factor, platelet derived-growth factor, recombinant somatomedin-C, multiplication-stimulating activity) and insulin had no consistent effect on the cell growth in SF2 either in the presence or absence of hGH. Corticosterone (12.4-150 nM) was toxic to the Nb2-11C cells. SF2 could support the growth of Nb2-11C cells for at least 30 passages in the presence of 5 ng/ml hGH, and that of 2 spontaneously proliferating cell lines (Nb2-SP and Nb2-HSP) for the same length of time in the absence of lactogen. However, in all cases the growth rate in SF2 was lower than that seen in the presence of 10% HS. Long-term culture of Nb2-SP and Nb2-HSP cells in SF2 led to an increase of the growth rate with time. There was a change in the responsiveness of Nb2-SP cells to lactogens after long-term culture in SF2 which was only apparent in the presence of HS. After 10 passages in SF2, Nb2-11C cells showed no apparent changes in lactogen-induced growth response, cell phenotype, cell size, or binding capacity for [125I]hGH.

Investigations of rinderpest antibody in East African Wildlife, 1967-1971.

Buffaloes showed no material evidence of infection in W. Uganda (1/77 positive) but the disease probably occurred in E. Kenya in 1969/70. In the Serengeti area of Tanzania one batch of sera collected in August 1969 showed low antibody titre (10 or less) in 7 of 15 animals 2 to 5 years old. This activity was restricted both geographically and temporally, as well as being difficult to assay in tube neutralisation tests.

Seroepidemiological survey of the prevalence of antibodies to a strain of human calicivirus.

Batches of pooled immune globulins and sera were tested by immune electron microscopy (IEM) for the presence of antibodies to a strain of human calicivirus (HCV, UK1). The results show that this strain of HCV is prevalent throughout many parts of the world and that the majority of the population experience infection by the age of 12 years. The survey carried out in the United Kingdom indicates that the presence of maternal antibody correlates with some degree of protection during the first few weeks of life; the peak incidence of cases and acquisition of antibody occur between 3 months and 6 years. Tests on sera from Japan show a similar pattern of acquisition of antibodies and demonstrate that infection with more than one strain of HCV commonly occurs during childhood.

Maintenance of granulopoiesis in long-term bone marrow cultures from W/Wv mice and effects of lipopolysaccharide on granulopoiesis in culture.

An attempt was made to establish long-term cultures of marrow cells from genetically anaemic W/Wv mice. Two batches of horse sera were used. One batch of horse serum (HS-lot A) supported long-term maintenance (up to 20 weeks) of granulopoiesis in vitro. The number of suspension cells in W/Wv marrow culture was maintained at the same level as that in the control +/+ culture, but the number of granulocyte-macrophage progenitor cells (GM-CFC) and the ratio of immature to mature granulocytes were at a lower level than those in +/+ culture. These data suggest that haemopoietic progenitors in W/Wv cultures maintain a higher level of differentiation, and hence an increased self-renewal than those in +/+ cultures. Another batch of horse serum (HS-lot B) was less effective in the maintenance of the cultures, and the cultures deteriorated within 10 weeks. Addition of bacterial lipopolysaccharide (LPS) induced increased granulopoiesis in +/+ cultures, whereas such treatment resulted in the depletion of suspension cells in W/Wv cultures. The results suggest that haemopoietic cells of W/Wv mouse cannot cope with the strong stimulus for differentiation that occurs after the administration of LPS, although the cells can continue a moderately increased self-renewal and differentiation, as indicated by the results in the culture with HS-lot A.

The effect of cell passage on the susceptibility of BALB/3T3 clone A31-1-1 cells to 3-methylcholanthrene-induced morphological transformation.

The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO2 incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14 had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.