Spectrophotometric quantitation of anchorage-dependent cell numbers using the bicinchoninic acid protein assay reagent

A rapid and convenient method is described for the determination of the actual and relative number of adherent cells in tissue culture. The cell lines human melanoma C32, ATCC CRL 1585, mouse melanoma B16-F10, and pig epithelial LLC-PK1, suspended in Dulbecco's minimum essential medium containing no serum, were allowed to adhere to fibronectin adsorbed to wells of a 96-well microtiter plate. Nonadherent cells were removed by aspiration, wells were washed, and adherent cells were solubilized with 200 microliters of the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay reagent. Plates were heated to 60 degrees C for 30 min and absorbances read at 562 nm using a microtiter plate reader. A linear correlation was observed between the number of adherent cells in the range 2-8 X 10(5)/ml cells added and the protein content of the adherent cells as measured by the BCA protein reagent. The assay procedure gave absorbance values in the range of 0.100 to 1.30 making the method highly sensitive and reproducible. Blank wells containing only coupled protein and no cells gave little or no absorbance. Cell adhesion was fibronectin specific since little or no cell attachment was observed when microtiter plates were coupled with bovine serum albumin. Similar results were obtained with other cell types such as platelets. These results indicate that measurement of total cellular protein using the BCA protein reagent can be a rapid and sensitive assay for the detection and quantitation of adherent cells.     

Measurement of the specific lipid content of attached cells in microtiter cultures

A method has been described to quantify intracellular neutral lipid content in attached cells in microtiter cultures. The procedure was based on oil red O staining of neutral lipid and Coomassie brilliant blue G-250 staining of total cellular protein. Results were expressed as the ratio of lipid to protein, the "specific lipid content" index. This measurement was shown to closely correspond to actual lipid per cell measurements under experimental conditions. The procedure was specific for neutral lipids and sensitive (greater than or equal to 50 ng triglyceride/well). Additionally, cell proliferation measurements could be made simultaneously, using protein staining data. Chromatic endpoints were measured using a spectrophotometer capable of reading individual wells of a microtiter plate. The procedure is recommended for applications in which the endpoint is neutral lipid droplet accumulation in attached cultured cells.     

A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells

A solid-phase radioimmunoassay to detect antibodies that react with antigens derived from human melanoma cells is described. A soluble preparation derived from Nonidet P-40 lysates of tissue-cultured melanoma cells was dried on the surfaces of wells of polyvinyl chloride microtiter plates and fixed with 0.02% glutaraldehyde. Antibody preparations were added and incubated for 18 h at 4 degrees C. The wells were washed and bound antibodies were detected using radioactive Staphyloccoccal protein A (125I-SpA). Optimal conditions are described for all the steps employed. Concentrations of antigen selected, the amount of 125I-SpA employed and the duration of incubation of antibodies with antigen were found to be critical. The assay was sensitive and reproducible, and lent itself to the simultaneous evaluation of many individual antibody samples in a short period of time. The assay was particularly valuable for rapid screening of hybridoma supernatants for antibodies to antigens derived from melanoma cells and from a panel of other tumor and normal cells.     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.     

An automated method for DNA preparation from thousands of YAC clones.

We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library. Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate. The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR). Using this protocol, we produced 3000 YAC strain DNAs in three weeks. This automated procedure should be extremely useful in many genomic mapping projects.     

Neural cell adhesion molecule expression is regulated by Schwann cell- neuron interactions in culture

To investigate the cellular and molecular signals underlying regulation of cell adhesion molecule expression, the influence of interactions between dorsal root ganglion neurons and Schwann cells on their expression of L1 and N-CAM was quantitated by immunogold electronmicroscopy. The numbers of antibody binding sites on cell surfaces of neurons and glia were compared between pure populations and co-cultures. After 3 d of co-culture, expression of L1 was reduced by 91% on Schwann cells and 36% on neurons, with expression in pure cultures being taken as 100%. N-CAM expression was unchanged on neurons and reduced by 43% on Schwann cells. Within 3 d after removal of neurons from Schwann cell-neuron co-cultures by immunocytolysis, expression of L1 and N-CAM on Schwann cell surfaces increased by 69 and 84%, respectively. Cell surface antigens recognized by an antibody to mouse liver membranes were unchanged in co-cultures. Furthermore, in co-cultures of neurons and sciatic nerve fibroblasts neither of the three antibodies detected any changes in expression of antigens when pure and co-cultures were compared. These observations suggest that adhesion molecules are not only involved in neuron-Schwann cell recognition and neurite outgrowth on Schwann cells (Seilheimer, B., and M. Schachner. 1988. J. Cell Biol. 107: 341-351), but that cell interactions, in turn, modulate the extent of adhesion molecule expression.     

Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein

A method has been developed to determine the relative or actual number of attached cells in microtiter plate wells without making direct cell counts. The procedure is based upon staining total cellular protein with Coomassie brilliant blue G-250, followed by measurement of absorbance at 630 nm in a spectrophotometer designed to read each well of a 96-well microtiter plate. No destaining of cells is required. A linear correlation exists (r = 0.970) between cell number and absorbance over a useful range. Intraplate well-to-well variation is acceptable (CV = 0.101). This method was used to measure the proliferative response of human vascular smooth muscle cells to human serum. It should be useful in other assays involving proliferation of attached cultured cells.     

Survival of human lymphoblastoid cells after DNA damage measured by growth in microtiter wells

Survival of cells in suspension culture after treatment with damaging agents is usually measured by extrapolation from growth curves or by growth of colonies in soft agar. We have developed a survival assay which measures the ability of small numbers of cells to initiate microscopic cultures in wells of microtiter plates without agar or feeder layers. Suitable human lymphoblastoid lines were obtained by selection of rapidly growing cultures from microtiter wells in which <200 cells were inoculated in 0.2 ml RPMI 1640 medium and incubated at 37° with 5% CO₂ at 95% relative humidity. Survival after damage was measured by inoculating groups of 24 microtiter wells with appropriate serial dilutions of cells. The wells were examined microscopically at intervals and scored for evidence of cell proliferation. Survival was calculated with the Poisson formula on the basis of the fraction of wells in which cells were not proliferating. Survival did not change appeciably after 2–3 weeks incubation. Survival measured by the microtiter-well assay was found to be similar to survival measured by extrapolation from growth curves after damaging the cells with bleomycin or with 8-methoxypsoralen plus long-wavelength ultraviolet radiation. The microtiter-well assay affords a simple, accurate measure of cell survival in human lymphoblastoid cells with suitable growth ability.     

A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general.     

表达β-半乳糖苷酶的gal B16肿瘤模型的建立及其在肿瘤DNA疫苗研究中的应用

本文工作的目的是建立以β-半乳糖苷酶为标志性抗原的小鼠黑色素瘤模型,并进行肿瘤免疫的研究。我们首先在pcDNA3质粒中引入一个β-半乳糖苷酶编码基因从而建立转染质粒p3gal。p3gal转染小鼠黑色素瘤细胞B16后,再通过G418筛选及X-Gal细胞染色得到表达β-半乳糖苷酶的gal B16细胞株。接着用该细胞株成功地在C57小鼠上建立了表达β-半乳糖苷酶的gal B16肿瘤模型。并在此模型上观察了β-半乳糖苷酶编码基因作为DNA疫苗抑制gal B16肿瘤生长的作用。     

The role of inhibitors in the fluorescent staining of benign naevus and malignant melanoma cells with 9-amino acridine and acridine orange

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

The Role of Inhibitors in the Fluorescent Staining of Benign Naevus and Malignant Melanoma Cells with 9-Amino Acridine and Acridine Orange

Abstract

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence-activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouse-IgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.     

T cell immunotherapy for melanoma from bedside to bench to barn and back: how conceptual advances in experimental mouse models can be translated into clinical benefit for patients

A solid scientific basis now supports the concept that cytotoxic T lymphocytes can specifically recognize and destroy melanoma cells. Over the last decades, clinicians and basic scientists have joined forces to advance our concepts of melanoma immunobiology. This has catalyzed the rational development of therapeutic approaches to enforce melanoma‐specific T cell responses. Preclinical studies in experimental mouse models paved the way for their successful translation into clinical benefit for patients with metastatic melanoma. A more thorough understanding of how melanomas develop resistance to T cell immunotherapy is necessary to extend this success. This requires a continued interdisciplinary effort of melanoma biologists and immunologists that closely connects clinical observations with in vitro investigations and appropriate in vivo mouse models: From bedside to bench to barn and back.     

Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes

We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.     

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.     

Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.     

Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.     

小鼠主动脉血管平滑肌原代细胞的分离培养及鉴定

目的:血管平滑肌细胞在人类心血管疾病中具有重要的作用,而作为重要的遗传学研究模式生物的小鼠血管平滑肌材料有限,因此建立一种简单高效的小鼠血管平滑肌原代细胞分离培养方法很重要。方法:分离小鼠主动脉中膜层,胶原酶消化法获得原代平滑肌细胞,免疫荧光方法检测细胞的纯度和分化状态;分离平滑肌细胞特异的报告小鼠的平滑肌细胞,LacZ染色鉴定。结果:用该方法分离的原代平滑肌细胞生长迅速,3d后即可达5×106个。免疫荧光显示,细胞传至第3代后纯度在98%以上,细胞传至8代分化状态没有改变。LacZ染色鉴定报告小鼠分离的3代平滑肌细胞98%以上显示特异的蓝染。两种实验证明,应用此方法分离原代平滑肌细胞可以满足平滑肌体外功能实验的需求。结论:与传统的组织块培养法相比,该方法操作简便、经济,可以获得更多高纯度的血管平滑肌细胞。