Cell surface and substrate distribution of the 67-kDa laminin-binding protein determined by using a ligand photoaffinity probe.

BACKGROUND: Peptide 11, a nine-amino acid sequence from the beta1 chain of laminin-1, has been reported to inhibit tumor cell invasion of basement membranes, and to reduce tumor lung colonization (Iwamoto et al.: Science 238:1132-1134, 1987; Landowski et al.: Clin Exp Metastasis 13:357-372, 1995). The peptide is a ligand for the 32/67-kDa laminin-binding protein (LBP); however, the mechanism by which the 67-kDa LBP promotes invasion is unknown. METHODS: We have synthesized a highly specific probe for the 67-kDa LBP by adding a biotinylated residue, and replacing the required tyrosine in peptide 11 with the photoactivatable bezophenone crosslinker, 4-benzoyl-L-phenylalanine. This probe was used to follow the distribution of the 67-kDa LBP by gel electrophoresis, fluorescence-activated cell scanning, and confocal microscopy techniques. RESULTS: A single crosslinked protein, consistent with the high molecular weight form of the LBP, was found on Western blots of membrane detergent extracts from cells treated with the ligand probe. A CHO cell line, manipulated to overexpress the laminin-specific alpha6beta1 integrin, exhibited increased invasiveness, and expressed more cell surface 67-kDa LBP. Membrane-associated 67-kDa LBP was found in the vicinity of focal adhesion plaques and also associated with the matrix substrate. Studies on conditioned medium indicated that the matrix-associated LBP derived from material that was shed from the cells, with more being shed from the more invasive CHO variants. CONCLUSIONS: These results demonstrate the utility of this novel probe in diverse experimental protocols, and suggest that shedding of the 67-kDa LBP may have a role in promoting tumor cell invasion.

Introduction of antibody into viable cells using electroporation.

Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.

Isolation of viable tumor cells following introduction of labelled antibody to an intracellular oncogene product using electroporation.

A method for labelling the intracellular ras oncogene product, p21, with a monoclonal antibody, in B16BL6 mouse melanoma cells for subsequent flow cytometric analysis and viable cell sorting is described. Permeabilization of the cells for introduction of labelled antibody was attempted using (1) lysolecithin treatment, and (2) electroporation, a much more highly controllable technique. Permeabilization was assessed using propidium iodide or calcofluor white M2R staining, while short-term cellular viability was determined using fluorescein diacetate staining and long-term viability by reculturing the sorted cells. We successfully introduced labelled antibody into the cells with both permeabilization techniques. Insufficient numbers of viable permeabilized cells were obtained lysolecithin treatment to warrant an attempt at viable cell sorting. On the other hand, good numbers of viable, permeabilized cells were obtained using electroporation and we successfully sorted viable tumor cell populations based on the intensity of their anti-p21ras staining. These sorted tumor cells retained their characteristic anti-p21ras staining intensity for at least 2 weeks of propagation in culture.

Flow cytometric measurement of pollutant stresses on algal cells.

The lichen Usnea fulvoreagens (Räs). Räs. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a "stress index" of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low "stress index" of FDA fluorescence.

A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R.

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: isolation of fixed neurophysin neurons.

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80-85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.