Indicator cells

Among interface capturing methods, one of the most popular and most successful schemes is the volume-of-fluid (VOF) method dating back to the work of Hirt and Nichols [174]. The VOF method is based on a volume-fraction field c, assuming values between 0 and 1. A value of c = 1 indicates cells that are filled with phase 1, and phase 2 corresponds to c= 0. Intermediate values of c indicate the position of the interface between the phases however, the goal is to maintain a sharp interface in order to identify the different fluid phases uniquely. Volumes assigned to the different phases are moving with the local flow velocity W , and therefore the evolution of c is determined by a convection equation ... 

By definition, cell mitosis results in two daughter cells, identical to each other and to the parent cell. So in order to break symmetry, bit 1 in the state vector is set to 1 in one cell, while it is set to the reverse (0) in the other cell. The first 3 bits of the state vector are prespecified as the ones indicating cell type — in reverse. For example, a cell with state vector = 001110 has type 001-reversed, which is 100 or 4 (in decimal). This entails that the first division inevitably results in two cells of different types with state vectors 000100 and 100100, respectively. 

TIRFM was used for time-lapse observations of initial cell adhesion to SAMs with different surface functionalities (Fig. 2). After 10 min of plating a suspension of human umbilical vein endothelial cells (HUVECs), a few bright spots were observed on SAMs with COOH and NH2 functionalities this indicated cell adherence. The number of bright spots increased and the spot areas enlarged with incubation time, indicating that HUVECs adhered and spread well on COOH-SAM and NH2-SAM surfaces. Quantitative analysis of the number of adherent cells and cell adhesion areas... 

Cell-free systems, when supplemented with relevant cofactors, are remarkably proficient, despite their crudity in generating reactive electrophiles from most procarcinogens. However, they provide at best a broad approximation of in vivo metabolism and can fail to produce sufficient quantity of a particular reactive metabolite to be detectable by the indicator cells or they can produce inappropriate metabolites that do not play a role in vivo (see Gatehouse and Tweats, 1987, for discussion). 

Preliminary Cytotoxicity Testing. An essential first step is to carry out a preliminary study to evaluate the toxicity of the test material to the indicator cells, under the conditions of the main mutagenicity test. When selecting dose levels, the solubility of the test compound, the resulting pH of the media, and the osmolality of the test solutions all need to be considered. The latter two parameters have been known to induce false positive effects in in vitro mammalian tests (Brusick, 1986). The experimental procedure is carried out as follows. 

For most of the supernatants tested the number of wild type Myl-D7 indicator cells decreased rapidly to less than 10% within the first 10 days. These mutants were classified as non secretors. Only 26 of the total 89 mutants secreted an activity stimulating the survivaFproliferation of Myl-D7 for at least two weeks. Furthermore, only ten of these secretor mutants (e.g. 6i-4, 6i-22) stimulated the growth of Myl-D7 for longer than 6 weeks (data not shown Heberlein et al, 2006). 

Human Lymphoblasts Versatile Indicator Cells for Many Forms of Chemically Induced Genetic Damage... 

Product Protein or MAb/immunogen Indication Cells First approval Coimtries... 

Fig. 9.1. XTH-2 cells derived from Xenopus tadpole heart endothelia, on polystyrene, 0.9 GHz (a) in culture medium (b) after 75 s in CD (cytochalasin D, 2 pg ml-1) (c) after 10 min in CD (d) after 15 min in CD-free culture medium again. In (a), the white arrows indicate bright areas where the cells are not in close contact with supporting surface in (b) the white arrows indicate cell nuclei, which are not visible in (a), and the black arrows indicate deviations of the interference fringe towards the periphery of a cell compared with (a) (Bereiter-Hahn 1987).

The particular cell line used in our laboratory, AHH-1 TK-(-/- cells (Crespi and Thilly, 1984), is a versatile indicator cell line for a variety of in vitro toxicological end points (Crespi et al., 1993 Crofton-Sleigh et al.,... 

From c. 1900 experiments, many of them with mammalian erythrocytes, indicated cells were selectively permeable to a range of low molecular weight neutral solutes. If molecules with similar structures were added to the suspension medium, entry of the test solute might be reduced. 

A two-color dot plot (Fig. 8.23) of cells stained with propidium iodide and annexin V FITC will indicate cells in three of the four quadrants. Unstained cells are alive and well and are the double negatives they neither express phosphatidylserine on their surface nor take up propidium iodide through leaky membranes. Cells that stain just with annexin V are apoptotic they have begun to express phosphatidylserine on their surface, but have not yet gone through the process that leads to permeabilization of their cytoplasmic membrane. Cells that stain both with propidium iodide and annexin V are necrotic (that is, dead) they take up propidium iodide and also stain with annexin Y. With a permeable cell, the flow cytometer cannot tell us whether the annexin V is on the outside of the membrane (because the cells have gone through apoptosis before membrane permeabilization) or on the inside of the membrane (because the cells have died by the necrotic pathway without apoptosis). 

If excessive cytotoxicity to the indicator cell cultures was caused by the test substance or suspected, the affected dilution(s) may be passed through individual Sepha-dex gel filtration columns following titration to aid in reducing the toxicity. In such a case identical dilutions of the controls must also be passed through individual columns. 

The RMK cell line, which exhibits cytopathic effect (CPE) in the presence of Influenza A(H1N1) or Avian Influenza A (H3N2) virus, was used as the indicator cell line in the infectivity assays. Cells in multiwell culture dishes were inoculated in quadruplicate with 0.1 ml of the dilutions prepared from test and control groups. Uninfected indicator cell cultures (cell controls) were inoculated with test medium alone. The cultures were incubated at 36-38° C. in a humidified atmosphere of 5-7% C02 in sterile disposable cell culture labware. The cultures were scored periodically for approximately seven days for the absence or presence of CPE, cytotoxicity and for viability. 

Figure 5.8 The crystal structure of olivine, (a) The structure projected onto (100) showing serrated chains of octahedra running parallel to the c axis (b) oxygen coordination polyhedra projected about the Ml and M2 positions. Metal—oxygen distances in each coordination site are indicated. Cell parameters and interatomic distances are for fay-alite (from Smyth Bish, 1988).

Difficulty is encountered in detecting mycoplasma in certain cell lines and, for this reason, indicator cells e.g. mouse 3T3 cells are often used. 24 hours after establishing a cover slip culture of 3T3 cells a sample of the supernatant of the cells under test is added to the culture. Positive and negative controls should also be included. Three to four days later cultures are tested using fluorescence staining and another technique. 

Fig. 5. Increased expression of human GM-CSF by immobilized plant cells [76]. Closed symbols indicate normal batch production and open symbols indicate cells encapsulated by alginate. The effects of encapsulation increase the concentration of extracellular protein...

The PEC cell s current voltage characteristic, which indicates cell performance, is shown in Figure 15.3 [10]. Twenty years ago the short lifetime of the PEC cells... 

Fig. 11.6. Icmt inhibition impairs activation of RhoA and Racl GTPases and impacts on RhoA- and Racl-mediated cell migration. A, thrombin-mediated activation of RhoA. Cells were treated with thrombin (-f) or vehicle (—) for 15 min, whereupon levels of bound and total RhoA were determined. B, EGF-mediated activation of Racl. Cells were treated with EGF (-f) or vehicle (—) for 15 min, whereupon lysates were prepared and levels of bound and total Racl were determined. C and D, RhoA rescues directed migration. MDA-MB-231 cells were treated for 3 days with cysmethynil Cysmeth) or vehicle Cont) and recombinant adenovirus carrying HA-tagged RhoA or GFP was introduced as indicated. Cells were harvested and transwell migration assays were conducted. E and F, Racl rescues random migration. MDA-MB-231 cells were treated as described above, except that recombinant adenovirus carrying HA-tagged Racl or GFP was introduced. This figure is reproduced from Ref. [28].

Lleonart, R., Naf, D., Browning, H., and Weissman, C. (1990). A novel, quantitative bioassay for type 1 interferon using a recombinant indicator cell line. Bio/Technology 8,1263 1267. 

Fig. 21 Evidence of successful gene transfection with bacterial P-galactosidase vectors with IL in hypoxic cells, or with cationic liposomes or IgG-liposomes. The P-galactosidase activity was developed with X-Gal at pH 7.0. Cells treated with IL-P-galacto-sidase vector showed many cells with blue color. The culture was also confluent. Cells treated with cationic liposome-P-galactosidase vector showed only two cells with gene expression. The cells were also not confluent indicating cell attrition. IgG-liposome treated cells also showed no P-galactosidase expression.

A recommended indicator cell line is the Vero African green monkey kidney cell line, which has a high cytoplasm/nucleus ratio. The indicator cells are added at a concentration of approximately 1 x 10" cells ml to sterile coverslips in tissue culture dishes 10-24 h before inoculation. The test sample should be added at a concentration of approximately 5 x 10 cells ml" to give a semi-confluent mono-layer at the time of observation (at 1 and 3 days post-inoculation of the sample). Slides are prepared in the same way as those prepared by the direct method. 

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