Pretreatment of cells

Cells are subjected to a standard pretreatment to ensure that they are in exponential phase for experiments. Pretreat cells for 48 h prior to any experiment by plating in 75-cm flasks and feeding with fresh medium 24 h before use. Determine the seeding concentration for each cell line examples are 1 x 10 cells per flask for NRK cells and 2.5 x 10 cells per flask for Hep-2 cells. 

Cell and Tissue Culture Laboratory Procedures in Biotechnology, edited by A. Doyle and J.B. Griffiths. 1998 John Wiley Sons Ltd. 

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As already mentioned, van Kuijk and colleagues (Kalariya et al., 2008) tested the effects of oxidation products of [i-carotcnc, lutein, and zeaxanthin on the activation of redox-sensitive transcription factors, NF-kB, and AP-1 in cultured ARPE-19 cells. Degradation products of all three carotenoids induced activation of NF-kB and AP-1, and these effects were ameliorated by pretreatment of cells with 1 mM NAC. NF-kB is a major transcription factor that binds to promoter sites of many pro-inflammatory cytokines such as IL-1, IL-6, TNF-a, and iNOS. These results indicate that the degradation products of carotenoids can stimulate a pro-inflammatory pathway. 

GM-CSF and IL-3 have been shown to compete for receptors in some types of cells (e.g. eosinophils and KG-1 cells), indicating some structural homology between GM-CSF and IL-3 receptors, perhaps because they share certain subunits or adapter proteins. GM-CSF occupancy results in phosphorylation of certain proteins, and because the receptor possesses no inherent kinase activity, receptor occupancy must be linked to kinase activity via the generation of second messenger molecules. Pretreatment of cells with pertussis toxin abolishes the effects of GM-CSF, indicating the involvement of G-proteins in signal transduction. Priming of neutrophil functions with GM-CSF involves the activation of phospholipases A2 and D. 

Cell suspension cultures of Gypsophila paniculata and Saponaria officinalis produce very closely related triterpenoid saponins. Pretreatment of cell suspension cultures of G. paniculata with gypsogenin 3,0-glucuxonide (a triterpenoid saponin precursor in G. paniculata) followed by administration of [ C] acetate resulted in a marked reduction in incorporation of radioactivity into saponins and their precursors, but not into sterols and steryl glycosides [26]. Measurements of OSC activities revealed that there was no effect of elicitor treatment on CS levels in either species, but in G. paniculata AS levels went down while in S. officinalis they increased. This suggests that in these two species OSCs are regulating steps in the isoprenoid pathway and control the flux to sterols and triter-penes. 

Multiple in situ nucleic acid detection can be achieved in two ways- by sequential hybridization with probes labeled with the same reporter and by simultaneous hybridization using probes labeled with different reporters. Any of the reporters listed above can be combined to allow dual nucleic acid detection, but we have found biotm and digoxigenin to be the most generally useful, since they are safe and sensitive Probes labeled with these reporters can be combined m NISH and detected differentially according to the scheme presented m Fig. 1 (6) 2. Pretreatment of cells/tissues Having chosen the reporter and incorporated it into a probe, the cell/tissue to be analyzed is prepared for hybridization The following requirements must be met to allow a successful hybridization reaction to take place. 

Pretreatment of cell membranes with cholera toxin specifically blocked the membrane GM1-ganglioside from reacting with galactose oxidase (13). 

The inhibitory effects of tea polyphenols on UVB-induced phosphatidylinositol-3-kinase (P13K) activation has been demonstrated in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with EGCG and TF-3 inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70S6 kinase (p °S6-K), PI3K downstream effectors, were also attenuated by these tea polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with MAP-... 

NISH technology has four elements (1) choice of probe/reporter and probe labeling, (2) pretreatment of cells/tissues, (3) denaturation/hybrid-ization, and (4) detection of signal. 

Into each tube, transfer directly 10 jxl of controls or cell suspensions to be tested under the mineral oil (no pretreatment of cell suspension is required). 

The two ceil lines differ in sensitivity to test compounds mou.se cell line much more resistant to carboxvlesterase inhibition hy ail compounds cell line profiles differ for each esterase carboxy[esterase is less sensitive than AChE and NTE to OP-induced inhibition pretreatment of cells with poor inhibitors of carboxvlesterase followed by treatment with strong inhibitors (paraoxon or malaoxon) is additive, suggesting that carboxvlesterase does not decrease OP compounds available for AChE inhibition. 

Figure 1 Uptake of PLGA nanoparticles containing tetramethylrhodamine-labeled dextran by monocyte-derived human immature DCs in culture. DC surface was labeled FITC-labeled concanavalin and shows veiling on the cell membrance (A). Pretreatment of cells with cytochalasin B resulted in inhibition of the nanoparticle uptake (C) whereas placebo treated cells showed strong uptake (B). ) (From Ref 125.)...

Fite and colleagues (56,57) investigated the possibility that fatty acids, in particular CLA, may enhance the therapeutic potential of docetaxel in breast eancer cells. Breast cancer cells were treated concurrently and sequentially with 40 pmol/L of CLA mix (50 50 9,11 10,12), c9,tll and il0,cl2 isoforms in combination with docetaxel at concentrations of 0-64 pmol/L for 48 h. The MTT assay was used to determine cell viability and the 50% inhibitory concentration (ICjq) of docetaxel determined in the presence and absence of individual fatty acids. Cell growth was inhibited in MCF-7 and MBA-MB-231 cancer cells by treatment with the il0,cl2 isoform but not the c9,tl 1 isoform or the parent linoleic acid. The ICjq values for docetaxel were reduced in both cell lines by treatment with CLA with the exception of c9,ill CLA in MCF-7 cells, which had no effect. Pretreatment of cells with all the different forms of CLA before docetaxel exposure also reduced IC50 values. These findings indicate that the presence of CLA can enhance the antitumor effects of docetaxel in breast cancer cells. This has important implications for cancer chemotherapy and possible reduction of side effects of standard therapies. 

In more recent studies, the effect of ascorbate was investigated in latently infected cell lines that had been stimulated with tumor promoter or inflammatory cytokine to trigger virus production (Harakeh and Jariwalla, 1994, and unpublished data). Pretreatment of cells with 100-300 jLg/ml ascorbate followed by cell stimulation with phorbol ester (PMA) or cytokine (TNF-a) resulted in dose-dependent suppression of virus activation. Unlike N-acetyl cysteine (NAC), which suppressed cytokine-stimulated HIV expression through inhibition of transcriptional activation by NF-kB, ascorbate seemed to have no effect on the activity of this transcription factor. AZT, a known inhibitor of de novo infection, had no effect on virus production in either unstimulated chronically infected cells or in stimulated latently infected cells (Harakeh and Jariwalla, 1994). 

Cd(II) can bind to both the bases and the phosphate groups of DNA, and tends to destabilize the DNA helix (Jacobson and Turner 1980). However, these effects do not constitute DNA damage in the usual sense of the term (i.e., strand breaks, cross-links, adducts). Incubation of DNA with Cd(II) alone or with H2O2 did not result in strand breaks (Roy and Rossman, unpublished data). Cadmium-metallothionein complex is able to induce DNA strand breaks (Muller et al. 1991). However, the production of DNA strand breaks in cells by a cadmium-metallothionein complex is unlikely. Pretreatment of cells with Zn(II) to induce metallothionein resulted in decreased toxicity and DNA strand breaks by Cd(II) (Coogan et al. 1992). Transfection of a metallothionein overproducing plasmid into G12 cells abolished the mutagenicity of Cd(II) (Goncharova and Rossman, in preparation). 

Flavonoid pretreatment of cells may act to prevent the peroxidation of cellular membrane lipids. For example, the preincubation of cultured retinal cells with eriodictyol, luteolin, quercetin, and taxifolin evoked protection against Fe " ascorbate-induced lipid peroxidation and increases in intracellular oxidative stress... 

On the basis of such pretreatments of cells, four systems of transfection can be distinguished. Three of these are schematically depicted in Fig. 1. 

Pretreatment of cells with various agents, homogenization, and gradient centrifugation Homogenization and isopycnic centrifugation... 

Deuterium oxide was known to stabilize microtubules and, when applied to pancreatic endocrine cells, resulted in a dose-dependent inhibition of secretion which eventually reached complete cessation of insulin release. Pretreatment of cells with D2O protected them from the effects of colchicine or vinblastine when D2O was removed from the incubation media. Similarly, pretreatment of cells with cytocholasin B protected the cells from the effects of D2O. 

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