Biomedical subjects

J L Vierck

Publications and source records attributed to J L Vierck.

9 recordsLinked to original sources

PubMed2005

Gaining a solid grip on adipogenesis.

Obesity is presently being combated by fitness regimens, drugs and diet. Increasing our understanding of the physiology of adipocytes, by deducing the regulatory pathways involved in lipid metabolism and all aspects of adipogenesis, will provide alternative strategies to reduce adverse problems of obesity. Research has suggested that mature fat cells may dedifferentiate to form proliferative-competent fat cell precursors. Knowledge of the dedifferentiation process will allow us to gain a solid grip on adipogenesis.

Adipocytes↗

PubMed2001

Patterns of expression of muscle-specific markers of differentiation in satellite cell cultures: determination by enzyme-linked immunoculture assay and confocal immunofluorescent assay.

Equine satellite cell clone SE-11 and ovine satellite cell clone I(1)were evaluated for expression of myosin heavy chain, myogenin, desmin, and muscle-specific actin over a 240 h period in culture. An enzyme-linked immunoculture assay (ELICA) was capable of detecting these proteins at all time points evaluated. A linear relationship was demonstrated between the natural logarithm of the absorbance values (corrected for cell number) from the ELICA and percent fusion in both SE-11 and I(1)cultures. The r(2)values for SE-11 cultures were: desmin 0.82, muscle actin 0.81, myogenin 0.78, and myosin 0.70. The r(2)values for I(1)cultures were: desmin 0.77, muscle actin 0.72, myogenin 0.70, and myosin 0.61. Our confocal results support the idea that differences exist between species in the differentiation dynamics of satellite cells. Further, these data suggest that the ELICA may be applied to previously conducted experiments, enabling additional data to be obtained with relation to muscle protein expression.

Actins↗

PubMed2000

Ten commandments for preventing contamination of primary cell cultures.

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive both in time and loss of materials. Through the consistent use of proper aseptic techniques, most instances of contamination may be avoided. We suggest that the basic principles detailed here will find wide applicability in the culturing of primary cells without contamination from many different types of animals and tissues.

Adipose Tissue↗

PubMed2000

Formulation of a defined medium to maintain cell health and viability in vitro.

The first step in formulating a defined medium is to conduct a thorough search of the scientific literature. If a defined medium formulation is located that might be compatible with the intended cell system, a pilot study should be carried out to evaluate the general performance of the medium. Depending on the initial data obtained from this study, individual components of the medium and their concentrations may need to be manipulated (added/subtracted, increased/decreased) to obtain the desired results. Also, sometimes the basal medium or proportions of basal media must be changed. Because the formulation of a defined medium is a circular process, alteration of the basal medium type or ratio of basal media will necessitate redoing all of the previous addition/subtraction and optimization steps. Revalidation must also be done if vendors of components are changed or whenever different cells or cells of other ages are used in the system. This paper presents a brief procedure for formulating a defined media and an overview of the application of two defined media in muscle cell culture.

Animals↗

PubMed2000

Traditional and emerging methods for analyzing cell activity in cell culture.

The selection of appropriate techniques to assay for markers of cell activity is important for obtaining optimal results in cell culture-based research. This paper is intended as a guide to many of the assays currently available and new techniques that have been recently introduced in the literature. This paper addresses both manual assay techniques, including the use of hemocytometers, phase contrast microscopy, cell staining, and the immunofluorescent antibody assay (IFA), and automated assays for cell activity, including stained optical density, proliferating cell nuclear antigen, creatine kinase assay, DNA quantification, electronic cell counting, flow cytometry, magnetic cell sorting, image analysis, chemiluminescence, radioisotope labeling, precursor incorporation, in-situ hybridization/ligand binding, and enzyme-linked immuno-culture assay (ELICA). Advantages/disadvantages and applicability of these assays to different areas of cell culture research are discussed, and guidelines for selecting an appropriate assay are suggested.

Cell Count↗

PubMed2000

Interpretation of cell culture phenomena.

This paper discusses the dilemma of interpreting unusual or abnormal phenomena seen in cell cultures and is not intended to address the statistical design of experiments. Problems that can be encountered when growing cells in experimental situations include low or decreasing cell numbers, abnormal cell morphology, microbial contamination, and detachment of the cell monolayer. If any of these situations occur, it is not realistic to proceed with data analysis until the problem is corrected. The best policy is to attempt to standardize all types of cultures used for analysis and to avoid using any cultures that display atypical characteristics.

Animals↗

PubMed2000

Evaluating dot and Western blots using image analysis and pixel quantification of electronic images.

Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. After the DB were developed and dried, the images were digitized using an HP Deskscan II flat bed scanner, exported into Image-Pro Plus and analyzed by taking the combined mean of 45 degrees and 135 degrees sample lines drawn through each dot. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-I were assessed by this method. In the second procedure, WB were scanned with a ScanJet 3c flat bed scanner and their backgrounds were clarified using Image-Pro Plus. A second image analysis program, Alpha Imager 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying alpha-Actinin on the WB. Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images.

Actinin↗

PubMed1997

The development and utility of a defined muscle and fat co-culture system.

The utility of co-culture systems in defining the interactions between two different cell types has been well documented in the literature but is a relatively new tool for use in the study of cells derived from normal muscle tissue from meat animals. The majority of myonuclei in postnatal skeletal muscle cells (myofibers) result from satellite cell proliferation, differentiation and fusion with existing myofibers. As such, satellite cell culture systems that mimic postnatal myofibers have great potential for delineating the process of growth and repair of muscle mass. Other ways to simulate the environment of postnatal myofibers might include the development of co-culture systems using satellite cells, or satellite cell-derived myotubes, and other mesodermal cells commonly found associated with muscle tissue in vivo. This brief review describes our initial efforts to develop a defined satellite cell and preadipocyte co-culture system. We provide useful information about defined media requirements and requirements for proper cell orientation and growth on two different growth planes. We present summary data to suggest that differences were found between members of the insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) families of polypeptides, when conditioned media samples were analyzed from co-cultures composed of 3T3-L1 preadipocytes and satellite cells (with different propensities to undergo morphological fusion to form multinucleated myotubes). We also provide information about potential problems to avoid when initiating and conducting co-culture experiments. Such a co-culture system has application in the study of human obesity and also in the regulation of fat deposition in meat animals.

Adipocytes↗

PubMed1996

Proliferation and differentiation of progeny of ovine unilocular fat cells (adipofibroblasts).

The responsiveness of progency of sheep-derived unilocular fat cells (adipofibroblasts) to dexamethasone, insulin, insulinlike growth factor I (IGF-I), growth hormone (GH), and basic fibroblst growth factor (FGF) was determined in a clonal culture system. Primary cultures of mature adipocytes were obtained from intermuscular adipose tissue (semimembranosus/semitendinosus seam depot) of sheep by ceiling culture techniques. Following degeneration of unilocular fat droplets and re-establishment of fibroblasticlike adipofibroblasts, all adipofibroblasts adhering to upper flask surfaces were collected and isolated away from fibroblasts (which had no multilocular vesicles) by Percoll gradient centrifugation. Progeny derived from a single adipofibroblast were isolated and tested for the ability to proliferate, differentiate, and accumulate lipids. Stock cultures of adipofibroblasts reached confluence in 5 d and were induced to differentiate from 7 to 9 d with dexamethasone-methyl isobutylxanthine-insulin (DMI). Incubation with insulin, IGF-I, GH, or FGF prior to confluence followed by induction with DMI produced no direct (priming) effect on subsequent differentiation. When substituted individually in place of DMI during the 2 d differentiation/induction period, all factors induced differentiation of cultured adipofibroblasts as determined by lipogenesis (P < .05) and lipoprotein lipase activity (P < .05). Thus, isolated adipofibroblasts from sheep muscle may be induced by hormones and growth factors to display mature adipocyte morphology in cell culture. Further definition of the adipofibroblast culture system may aid in the identification of mechanisms regulating adipocyte development in sheep skeletal muscle, as well as in the study of intercommunication between fat and muscle cells.

3T3 Cells↗