Live-cell experiments

Measurements of Applied Chemoattractant Stimulations To quantitatively measure an applied cAMP concentration in live cell experiments, Alexa 594 mixed was mixed with cAMP and imaged using a confocal microscope with a Z-axis resolution of 1-2 pm (rrrNote 1). The intensity change of Alexa 594 and cell... 

FRET imaging can monitor dynamic interactions between a FRET donor (ECFP) and an acceptor (EYFP), which are fused to the protein of interest, in live cell experiments. As an example, we show the FRET changes of a fluorescent biosensor for Ca +, came-leon, using the spectral detector of the Zeiss LSM 510 META in a time-lapse imaging experiment. Cameleon is a chimeric protein that consists of ECFP, the calmodulin-binding peptide M13... 

There have been encouraging results reported within the context of live cell experiments using FTIR. The collective demonstration of biochemical stability for different cell types (T-1, HeLa and fibroblasts) by the various research groups working within this field, together with the suggestion of Moss et that correction for water absorbance may not be necessary, suggests that future FTIR studies may be able to measure early biochemical responses of... 

Figure 3, Basic concept of intracellular SERS probes Gold nanoparticles are transferred into cells. Aggregates which provide optimum SERS enhancement and are typically utilized in the live cell experiments are shown in the transmission electron micrograph and the schematic drawing. During excitation with laser light in the near-infrared (h VjJ, such gold nanoaggregates provide enhanced local optical fields in their nm-scaled vicinity, leading to surface-enhanced Stokes (h and anti Stokes...

Imaging of live OCCs requires a microscope stage incubator. To perform live cell experiments start by filling the medium reservoir of the microscope stage incubation chamber with culture medium. SrrNote 19. 

Biosynthesis of Protein. The dynamic equilibrium of body protein was confirmed by animal experiments using A/-labeled amino acids in 1939 (104). The human body is maintained by a continuous equilibrium between the biosynthesis of proteins and their degradative metabolism where the nitrogen lost as urea (about 85% of total excreted nitrogen) and other nitrogen compounds is about 12 g/d under ordinary conditions. The details of protein biosynthesis in living cells have been described (2,6) (see also Proteins). 

The first step in this study has involved experiments which synthesize alkaloids in vitro under quasi-cellular conditions, using reactions which can proceed in the living cell and compounds which actually occur in the cell or which are supposed to be intermediates in the plant metabolism. Such synthesesaredesignatedassyntheses under physiological conditions. ... 

A characteristic of all the above reactions is that the yield of the aldoliza-tion product depends on the pH of the reaction mixture (324), the maximum yield usually occurring near pH 7. Such reactions have been carried out in vitro in dilute aqueous buffer under so-called physiological conditions, i.e., conditions attainable in the living cell. Although this oversimplified technique for the study of alkaloid biogenesis is now being abandoned in favor of experiments in vivo with labeled precursors, such reactions are still of interest to organic chemists. 

The experimental results for hybridoma and protozoa cells given as examples in Fig. 25 indicate that much higher stress (4 to 30 times) is required under laminar flow conditions of viscosimeters than in stirred vessels to achieve the same death rate k. Here the death rate k is defined as first order deactivation constant k = 1/t In (Nq/N), where N, is the initial and N the time-dependent number of living cells in special deactivation experiments under otherwise optimal living conditions. The stress in Fig. 25 was calculated with Eq. (28) for stirred vessels and with Eq. (1) for the viscosimeter. Our own results for hybri-... 

This will extend, and partially replace, the traditional approach to biomedical research that is based on studying living cells or tissues in vitro, or on obtaining data from human volunteers in vivo, by introducing in silico experiments (a term, derived from the currently prevaihng sihcon-based computer chips). 

The cyan emitting form obtained by the mutation Y66W [10] deserves special attention, as it has led to the ECFP form (absorption at 437 nm, emission at 474 nm), whose variants are still intensively used as excitation energy donors in FRET imaging experiments for monitoring molecular associations in the living cell... 

The authors chose pyruvic acid as their model compound this C3 molecule plays a central role in the metabolism of living cells. It was recently synthesized for the first time under hydrothermal conditions (Cody et al., 2000). Hazen and Deamer carried out their experiments at pressures and temperatures similar to those in hydrothermal systems (but not chosen to simulate such systems). The non-enzymatic reactions, which took place in relatively concentrated aqueous solutions, were intended to identify the subsequent self-selection and self-organisation potential of prebiotic molecular species. A considerable series of complex organic molecules was tentatively identified, such as methoxy- or methyl-substituted methyl benzoates or 2, 3, 4-trimethyl-2-cyclopenten-l-one, to name only a few. In particular, polymerisation products of pyruvic acid, and products of consecutive reactions such as decarboxylation and cycloaddition, were observed the expected tar fraction was not found, but water-soluble components were found as well as a chloroform-soluble fraction. The latter showed similarities to chloroform-soluble compounds from the Murchison carbonaceous chondrite (Hazen and Deamer, 2007). 

The next question to address is to what extent does the study of a (deeply) frozen biological sample provide information that is relevant for an understanding of its functioning in a living cell at whatever the ambient temperature of this cell happens to be First and foremost, let us state the fact of experience that solutions of biomacromolecules such as metalloproteins can be frozen and thawed many times without any detectable deterioration of their biological activity. Combined with the rather low intensity (<0.2 W) of the microwave source of an EPR spectrometer, this leads to the proposition that EPR spectroscopy is a nondestructive technique. 

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