Cell culture

About: Cell culture is a research topic. Over the lifetime, 133361 publications have been published within this topic receiving 5364150 citations. The topic is also known as: cell culture techniques.

Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1.

Abstract: In an endeavor to further characterize human intercellular adhesion molecule-2 (ICAM-2), two murine monoclonal antibodies (mAb) were generated to ICAM-2 transfected COS cells, and designated CBR-IC2/1 and CBR-IC2/2. Immunoprecipitated, reduced ICAM-2 migrated as a broad band of Mr 60,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with N-glycanase revealed a peptide backbone of Mr 31,000, consistent with the size predicted from the cDNA. ICAM-2 had a broad distribution on hematopoietic cell lines and little expression on other cell lines, the sole exception being cultured endothelial cells which possess high levels of ICAM-2. Resting lymphocytes and monocytes expressed ICAM-2, while neutrophils did not. Staining of tissue sections with anti-ICAM-2 mAb confirmed their strong reactivity to vascular endothelium, but demonstrated a lack of ICAM-2 expression on other tissues. Small clusters of ICAM-2 positive cells were, however, seen in germinal centers. In contrast to ICAM-1 there was little or no induction of ICAM-2 expression on lymphocytes or cultured endothelium upon stimulation with inflammatory mediators. One of the two mAb, CBR-IC2/2, was found to totally inhibit binding of ICAM-2+ COS cells to purified lymphocyte function-associated antigen-1 (LFA-1). Using this mAb, LFA-1-dependent binding to both stimulated and unstimulated endothelium was found to be totally accounted for by ICAM-1 and ICAM-2. Homotypic aggregation of an Epstein-Barr virus-transformed B cell line, JY, was found to be solely ICAM-1 and ICAM-2-dependent, while in the case of the T cell lymphoma cell line, SKW3, anti- ICAM-2 mAb in conjunction with anti-ICAM-1 mAb could not inhibit the LFA-1-dependent aggregation. This suggests an additional LFA-1 ligand exists. Using a cell binding assay to purified LFA-1 in conjunction with anti-ICAM-1 and anti-ICAM-2 mAb, we have demonstrated that this putative third ligand for LFA-1 exists on SKW3 and other cell lines.

Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus

Abstract: The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory.

The nature and significance of osteopontin

Abstract: Osteopontin is an acidic glycoprotein of about 41,500 daltons that has been isolated from rat, human and bovine bone. It is rich in aspartic acid, glutamic acid and serine and contains about 30 monosaccharides, including 10 sialic acids. Several types of data suggest that the carbohydrate is present as 1 N-glycoside and 5-6 O-glycosides while the phosphate is present as 12 phosphoserines and 1 phosphothreonine. The cDNA sequence indicated the presence of a Gly-Arg-Gly-Asp-Ser- (GRGDS) amino acid sequence identical to a cell binding sequence in fibronectin, and suggested that osteopontin might function as a cell attachment factor. This conclusion is supported by a number of studies showing that the protein promotes attachment and spreading of fibroblasts and osteoblasts to substratum, and that this attachment is inhibited by RGD-containing peptides. Despite this evidence that it contains an RGD recognition sequence and probably interacts with the family of receptors known as integrins, it appears that osteopontin does not possess a collagen-binding domain. Osteopontin is synthesized by preosteoblasts, osteoblasts and osteocytes, is secreted into osteoid and is incorporated into bone. The expression at an early developmental stage is an indication that osteopontin is an important component in the formation of bone. The level of synthesis of osteopontin by osteoblasts in culture is increased by treating these cells with 1,25-dihydroxyvitamin D3 and TGF-beta. The effect of these agents is at the transcriptional level. In addition to bone cells, osteopontin is synthesized by extraosseous cells in the inner ear, brain, kidney, and deciduum and placenta. It is also synthesized by odontoblasts, certain bone marrow cells and hypertrophic chondrocytes. Studies with several fibroblast and epithelial-derived cell lines in culture indicate that secretion of osteopontin can be dramatically increased when these cells are treated with phorbol esters, growth factors and hormones. However, osteopontin does not appear to be expressed by mesenchymal cells, fibroblasts, epidermal cells or by most epithelial cells in vivo.

Growth Inhibition of Human Tumor Cells in Athymic Mice by Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies

Abstract: Monoclonal antibodies (MoAbs) were raised against epidermal growth factor (EGF) receptors on a human epidermoid carcinoma cell line, A431. Administration of anti-EGF receptor MoAbs inhibited tumor formation in athymic mice by A431 cells and by another epidermal carcinoma cell line, T222. When one of the same MoAbs was used in therapy against Li-7 (a human hepatoma) and HeLa cells (a cervical carcinoma), tumor growth was not affected. The number of EGF receptors on A431 cells was about 100-fold higher than on T222, Li-7, and HeLa cells, suggesting that the number of EGF receptors may not be an important determinant in suppressing tumor growth. Three anti-EGF receptor MoAbs were used in the present studies. MoAbs 528 (immunoglobulin G2a) and 225 (immunoglobulin G1) are capable of competing with EGF for receptor binding and inhibit proliferation of A431 cells in culture. The other MoAb, 455 (immunoglobulin G1), is incapable of blocking the binding of EGF to its receptors and has no effect on the proliferation of cultured A431 cells. All three MoAbs inhibited A431 tumor growth in athymic mice, indicating that the antibody isotype and the site of binding on the EGF receptor are not the determinants of antiproliferative activity in vivo. The observation that MoAb against the receptor for EGF is cytostatic rather than cytocidal in vitro against A431 cells, yet completely prevents tumor growth in vivo, suggests that some host animal responses also may be involved in the antitumor effect. MoAbs against growth factor receptors could provide useful immunotherapeutic agents.