Storage of chemotherapy drugs for use in the human tumor stem cell assay

Tumor chemosensitivity assays require the frequent in vitro use of antineoplastic drugs. Economy and convenience are greatly enhanced if these drugs are stored in aliquots for use as needed. This study investigated the stability of eight common antineoplastic agents at -60 degrees C. Activity was measured as inhibition of colony formation in a commonly used soft agar culture system using an experimentally induced murine sarcoma. Our results indicate no loss of activity with these drugs under the specified storage conditions.     

A simple and rapid assay for heme attachment to apocytochrome c

A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.     

A unique activity assay for carboxypeptidase A in human serum

Measurement of carboxypeptidase A, one of the pancreatic proteolytic enzymes, in human serum is made possible by a combination of affinity chromatography to isolate and concentrate the enzyme followed by monitoring activity spectrophotometrically with a high-turnover peptide substrate. Concentrations of enzyme in the nanogram-per-milliliter range can be determined with high precision and reliability. Initial clinical application of this method demonstrates no detectable activity in serum from normal individuals, but the enzyme is present in the sera of individuals with pancreatitis.     

cAMP binding protein assay for widespread use in cell signaling studies

cAMP plays a critical role in intracellular signaling pathways that regulate proliferation or differentiation. The cAMP binding protein assay, using a naturally derived cAMP binding protein, is one of the most widely used methods for cAMP determination. The major steps of this binding assay include purification of the binding protein, cAMP extraction from samples, and quantification of the cAMP Most purification methods of the cAMP binding protein were published before 1975, and many of the materials and methods are outdated. Here we describe an updated method of purification of cAMP binding protein from bovine skeletal muscle with the advantages of simplicity, low cost, and high yield The isolation procedures can be completed in two days using commercially available materials and equipment. The cAMP binding properties of the isolated protein can be utilizedfor more than two years. Binding protein isolatedfrom 1 kg bovine muscle is sufficientfor at least 3 x10(4) assay tubes. Furthemore, we describe the techniques of cAMP extraction and quantification that have been used successfully in studying parathyroid hormone signaling as an example of a G protein-linked seven transmembrane domain receptor that signals through the protein kinase A pathway.     

Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment

We have previously isolated mutants of the major-group human rhinovirus type 89 that grow in cells deficient in intercellular adhesion molecule 1 (ICAM-1), the receptor used by the wild-type virus for cell entry [A. Reischl, M. Reithmayer, G. Winsauer, R. Moser, I. Goesler, and D. Blaas., J. Virol. 75:9312-9319, 2001]. We now demonstrate that one of these variants utilizes heparan sulfate proteoglycan (HSPG) as a cellular receptor. Adaptation to ICAM-1-deficient cells not only resulted in the newly acquired receptor specificity but also rendered the virus less stable at low pH and at elevated temperatures. This instability might compensate for the absence of the uncoating activity of ICAM-1. Whereas wild-type virus infection via ICAM-1 proceeded in the presence of the vesicular H(+)-ATPase inhibitor bafilomycin A1, infection by the mutant via HSPG was prevented by the drug. This suggests that the low pH prevailing in endosomal compartments is required for uncoating in the absence of the catalytic activity of ICAM-1.     

Characterization of the KG1a cell line for use in a cell migration based screening assay

High-throughput screening has become a popular method used to identify new “leads” for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of variousin-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.     

A sensitive radiochemical assay for serum glutamic-oxaloacetic transaminase

A simple inexpensive hydraulic system for preparing uniform discontinuous gradients is described. The apparatus consists of five syringes, a three-way valve, plastic tubing, and three plastic rectangles. The apparatus is capable of delivering virtually any reasonable volume at any desired rate. It has been applied to the separation of platelets by centrifugation in discontinuous sucrose gradients.     

Preparation of delipidized serum protein for use in cell culture systems

Summary A rapid procedure for the preparation of delipidized serum protein is described. The delipidized protein can be used for the maintenance and growth of tissue culture cells in a lipid-free environment. The extraction procedure greatly reduces all serum lipid classes and the delipidized protein supports the growth of a variety of cells in culture. This research was supported in part by USPHS research grants HL-09103 and HL-16058 from the National Heart and Lung Institute and RR-05540 from the Division of Research Resources. This work was done during tenure as Established Investigator of the American Heart Association (G.H.R.).     

A rapid method for assay of 25-hydroxyvitamin D in serum

A simplified assay for 25-hydroxyvitamin D is reported. In this procedure ethanol extraction is used in order to obtain a better protein precipitation and a higher recovery of added tritiated 25-hydroxyvitamin D3. Extraction is followed by column chromatography on Sep-pak Silica cartridges. The eluate is dried and directly used for competitive protein binding assay. The method shows two main advantages: it is less time consuming and is easier to perform than existing procedures.     

A collaborative study of a preparation of normal human serum for use as a reference in the assay of beta 2 microglobulin

A biological standard for beta 2 microglobulin (beta 2m) determination has been prepared from pooled human serum by sampling and freeze-drying. A preliminary study of parallelism between the dose-response curves of the preparation and pure beta 2m or biological fluids was made with four different methods of assay and gave satisfactory results. In a collaborative study in five laboratories in five countries using a common method of assay, evidence was obtained that the preparation coded 80-12-3200 was suitable to serve as a standard for the assay of beta 2 microglobulin. Criteria included the constancy of the amount of material per vial and the stability of the freeze-dried product. The technique used for beta 2m determination was a radioimmunoassay.     

A cell culture assay for follicle-stimulating hormone

Cultured rat ovarian granulosa cells respond to follicle-stimulating hormone (FSH) by synthesizing and secreting plasminogen activator. The specificity of the response for FSH prompted us to explore the use of this system as an in vitro bioassay for FSH. The release of FSH by pituitary cell cultures has been examined by this method, as have been preparations of FSH of known biological activity. The results indicate that the granulosa cell system allows accurate, rapid, and convenient determinations of FSH activity. Furthermore, the method obviates metabolic clearance problems associated with whole animal assays and it is extremely sensitive: as little as 10(-15) mol (approximately 100 micronIU) of FSH can be detected.     

A semi-automated protein assay for cell cultures

A semi-automated modification of the protein determination procedure of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) is described. The assay is well suited to the analysis of the protein of adherent cultured cells. The procedure is carried out in 96-well microtest plates on protein solutions of 50 microliter or less, and can detect less than 0.5 micrograms of protein (equivalent to about 10(3) cultured cells). Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min.     

Routine heat inactivation of serum reduces its capacity to promote cell attachment

Summary Heat inactivation (56°C for 40 min) of bovine calf serum was shown to diminish its capacity to promote the attachment of cells to plastic or glass surfaces. This effect was not observed in stationary cultures (culture dishes) but became manifest under conditions in which the cells were subjected to a small amount of liquid shear force, i.e. by growing cells in roller bottles or culture tubes. Of four cell lines tested on bovine calf serum (SV-BHK, BALB-3T3, CV-1, and FS-4) SV-BHK and CV-1 cells showed the greatest sensitivity to loss of attachment-promoting activity. Fetal bovine serum also seemed to be affected by heat inactivation but to a lesser degree than bovine calf serum. Treatment of vessel surfaces with either unheated calf serum or specific attachment factors (gelatin, poly-d-lysine, and fibronectin) greatly increased cell attachment in the presence of heat inactivated serum. Heat inactivation did not seem to affect the ability of cells to grow after attachment. Of the four cell lines tested, the normal human fibroblast line (FS-4) was shown to be most effective at conditioning medium and restoring its capacity to promote the attachment of all four cell lines. This research was supported in part by VECOL, Inc., Bogata, Columbia and by grant SRC 5 U24 RR02557-02 from the National Institutes of Health, Bethesda, MD.