Experiment 1 Cells

FIGURE 11.3 One-way ANOVA (analysis of variance). One-way analysis of variance of basal rates of metabolism in melanophores (as measured by spontaneous dispersion of pigment due to G,.-protein activation) for four experiments. Cells were transiently transfected with cDNA for human calcitonin receptor (8 j-ig/ml) on four separate occasions to induce constitutive receptor activity. The means of the four basal readings for the cells for each experiment (see Table 11.4) are shown in the histogram (with standard errors). The one-way analysis of variance is used to determine whether there is a significant effect of test occasion (any one of the four experiments is different with respect to level of constitutive activity). 

Fig. 6.4 In vitro effects of mutation on desensitization and internalization of the dopamine receptor. Shown here are effects of mutation on dose-dependent intracellular cyclic adenosine monophosphate (cAMP) accumulation (A and B) and binding curves (C and D) for artificial ligand (SCH 23390) using three constructs controls (wild type, A and C) and the Thr360Ala mutant (360, B and D). In the desensitization experiments, cells were preincubated with 10 oA/ dopamine (o) or vehicle ( ) for 20min, and increasing concentrations of dopamine (10 to 10 (iM) were added to assess cAMP accumulation. Note that loss of efficacy and potency seen in wild-type cells (A) disappeared with the Thr360Ala mutation (B). Conversely, internalization, assessed by decrease in SCH23390 binding (C) after pretreatment with lOpM dopamine (o, compared to vehicle ), was essentially unchanged by the Thr360Ala mutation (D)...

Counterflow Experiments Cells are once again pre-loaded with a high concentration of unlabeled substrate. The cells are then placed in a medium containing a low concentration of labeled substrate or a low concentration of an alternative substrate which utilizes the same transport system. The investigator then measures the rate of entry of the labeled substrate or of the alternative substrate. 

Use the cell-flow technique (32) to determine the inhibition of the receptor. In these experiments, cells containing the uAChR are used. The maximum current amplitude, a measure of the concentration of open receptor-channels, is determined. The experiments are done in the presence of 100 pM carbamoylcholine (a stable analog of acetylcholine) and in the absence and presence of 150 pM cocaine. 

Cell cycle redistribution may not be the sole factor if cells are irradiated before drug exposure, but it has been shown that 5-FU can sensitize even when cells are irradiated before drug exposure. Byfield et al. found that 5-FU radiosensitizes HeLa cells only when the drug exposure followed radiation (the cells were treated with 5-FU, either before or after radiation, for up to 8 d). A similar finding was observed on HT29 human colon cancer cells, except that in these experiments cells were exposed to 5-FU for a maximum of only 30 min before radiation (33). These observations demonstrate that radiosensitization can be produced in the absence of cell cycle redistribution. 

Figure 4.7 — Absorption spectra of 2,4-dinitrophenylhydrazine (2,4-DNPH), 4-nitrophenylhydrazine (4-NPH) and 2-nitrophenylhydrazine (2-NPH) in solution (solid line) and retained on C bonded silica of 60-100 nm particle size (broken line). TTie dotted line corresponds to the blank spectrum in the second type of experiment (cell packed with C,j bonded silica in distilled water). (Reproduced from [97] with permission of Elsevier Science Publishers).

A semi-infinite transport geometry implies that, for the duration of the experiment, effects generated at the working electrode do not reach the other electrode or the cell walls, so that the transport coordinate behaves as if it were infinite in extent. The prefix semi reflects the fact that the geometry appears infinite in one direction (away from the electrode) but not in the other (towards the electrode). Because of the limited duration of most electrochemical experiments, cells may be tiny and yet be appropriately described as having a semi-infinite transport geometry. 

Depending upon the design of an experiment, cells were grown in 60 mm petri dishes, 150 cm2 plastic flasks (Falcon no. 3024), or roller bottles in MEM supplemented with 12% fetal bovine serum and antibiotics in 95% air, 5% C02 water humidified incubator at 37°. All experiments were initiated with confluent mono-layer of cells grown for about 5-15 generations. 

In similar experiments, cell-free preparations of a Streptomyces coelicolor strain containing the minimal PKS involved in actinorhodin biosynthesis were shown to produce compounds called SEK4 and SEK4b, molecules generated as shunt products of the normal pathway (Fig. 7). When the strain also included genes for the KR, ARO, and CYC activities, a more elaborate structure, 3,8-dihydroxy-l-methylanthraquinone-2-carboxylic acid (DMAC), was obtained (Fig. 7) [8], Biosynthesis of SEK4 and SEK4b has also been accomplished in... 

In these experiments cells are designed that have a thin electrolyte layer over the working electrode covered by a window that is transparent to the incident radiation. Refraction of the beam by the window has to be taken into account in the calculations. 

Fig. 7.2 Discontinuous cells used in the experiments - cell with rotating anode (left) and flowthrough cell with parallel plate electrodes (right). (1) Rotating contact, (2) electronically controlled stirrer, (3) thermostated beaker, (4) plate cathode with central hole or expanded mesh cathode, (5) rotating disk or expanded mesh anode, (6) parallel plate cell, (7) centrifugal pump, (8) stirred dark container for sampling, (9) magnetic stirrer, (10) cryostat...

Figure 13.27 (Top) Luminescence images of HeLa cells loaded with different concentrations of [Eu2(L62)3] in RPMI-1640 for 7h at 37°C. (Lex = 330nm, Xem >585nm, exposure time 60s). (Middle) Images of HeLa cells loaded with 250 p.M [Eu2(L62)3] (5h at 37°C, exposure time 10 s), then incubated with 40mgmL acridine orange (Xex = 450 90 nm Xem = 515-565 nm, exposure time 10 ms) in PBS (5 min at room temperature). (Bottom) Co-localization experiments cells loaded with 250 p.M [Eu2(L62)3] and 15 mgmL BIODIPY PL LDL (0.5 h, Xex = 470 nm, 2 s exposure time) [77]. (Reproduced from E. Deiters et al., Effect of the length of polyoxyethylene substituents on luminescent bimetallic lanthanide bioprobes, New Journal of Chemistry, 32, 1140-1152, 2008, by permission of The Royal Society of Chemistry (RSC) for the Centre National de la Recherche Scientifique (CNRS) and the RSC.)...

Figure 5.3 DNA flow cytometric analysis of cells treated with apigenin for 24 or 48 h. Each point represents the mean SD of three independent experiments. Cell cycle was monitored by a DNA flow cytometric analysis indicating as follows 0,% of G1-phase cells A,% of S-phase cells % of G2/M-phase cells. Means SD from three independent experiments are shown. p < 0.05 and p < 0.01 vs. the vehicle controls. (From Wang et at, Molecular Carcinogenesis, 28, 102-110, 2000. With permission.)...

Test cells (the same as source cells, if autocrine growth effects have to be shown) are plated at very low density (< 100 cells/cm ), in the same kind of medium used to produce the c.m. Once attached (for 4—8 h after plating), their medium is replaced with c.m. at different dilutions (100, 50, 10, 1 and 0%). Growth has to proceed for at least one week, so one change of medium can be done after 3-4 days. At the end of the experiment, cells can be quantitated by any of the methods described above. 

Cells are seeded in a microwell plate 2 to 3 days before the experiment to reach about 80 % confluency on the day of the experiment to favour adherence during subsequent manipulations. Remember that certain cell lines have to be cultured on a special matrix. On the day of the experiment cells in a microwell plate are transferred to a 37°C waterbath. The temperature should be checked with a thermometer since variations may influence exocytosis. Cells are first washed with KRH, than permeabilized with SLO in 0.1 iM free Ca ". The cells are exposed to SLO for 7 min. Subsequently, cells are preincubated (if necessary) at 0.1 xM Ca and then shifted to 0.1 M or 10 jxM Ca ". Finally, the supernatant is transferred to microcentrifuge tubes, centrifuged at 10 000 g for 2 min to sediment any contaminating cells thereafter aliquots are used for determination of the secretory product. 

For uptake experiments, cells are grown on 22-mm cover slips in six-well plates to 75-80% confluence. 

Methane oxidation kinetics was assessed as follows. In these experiments, cells were inoculated into medium containing different initial amounts of copper sulfate and grown to an optical density at 600 nm of 0.5-0.7. Aliquots were then placed in closed vials at different initial dissolved methane concentrations. These aliquots were incubated under optimal conditions, and head-space samples were taken at four different time points (1-4 h) for determination of methane concentrations by gas chromatography. From these data, initial methane consumption rates determined for different methane concentrations were used to obtain the Michaelis-Menten parameters Ks (half-saturation constant) and Vmax (rate at substrate saturation). Under these conditions sMMO was not expressed, as described previously (9). 

Bacteria that remained viable after 48 freeze-thaw cycles were used to initiate new cultures and these were subjected to additional freeze-thaw cycles as described. Individual isolates, which had been previously identified, as well as the control cultures, were also subjected to freeze-thaw cycles. Occasionally, single isolates in 10% TSB would supercool rather than freeze at temperatures close to 0°C, and to ensure that all samples froze at the same temperature, a few sterilized Agl crystals were added to the vials at the start of the experiments. For some experiments, cells were harvested by centrifugation (6,000 xg for 10 min) and the cell pellet washed with 10% TSB and kept at 0°C until analysis. Spent media was obtained by centrifuging, as above, and filtering (0.45 pm). 

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