Measurement of intracellular

A powerful tool now employed is that of diode array detection (DAD). This function allows peaks detected by UV to be scanned, and provides a spectral profile for each suspected microcystin. Microcystins have characteristic absorption profiles in the wavelength range 200-300 nm, and these can be used as an indication of identity without the concomitant use of purified microcystin standards for all variants. A HPLC-DAD analytical method has also been devised for measurement of intracellular and extracellular microcystins in water samples containing cyanobacteria. This method involves filtration of the cyanobacteria from the water sample. The cyanobacterial cells present on the filter are extracted with methanol and analysed by HPLC. The filtered water is subjected to solid-phase clean-up using C g cartridges, before elution with methanol and then HPLC analysis. 

Sheu, Y. A., Kricka, L. J., and Pritchett, D. B. (1993). Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells. Anal. Biochem. 209 343-347. 

Figure 8. Simultaneous measurement of intracellular Ca and oxidant production in neutrophils. Cells were labeled with Quin-2 and suspended at 2 x lo cells/mL buffer. At time zero, 1 nJf FLPEP was added (upper trace in each panel). In addition, the receptor blocker tBOC was added (3 x 10" M) after 30 s to stop further binding of the stimulus (lower trace in each panel). The excitation wavelength was 3A0 nm. Top panel Quin-2 fluorescence determined on channel B (of Figure 1) using a Corion A90-nm interference filter. The crossover from the superoxide assay has been subtracted. Middle panel Oxidant production (superoxide equivalents) determined by the para-hydroxyphenylacetate assay. Fluorescence was observed at AOO nm (on channel A of Figure 1).

FIG. 2 Measurements of intracellular potentials in sieve tubes of maize via severed aphid stylets. Stimulation by ice water (above) and electric shock (below) evoked action potentials which were propagated with a velocity of 3-5cms in a basipetal direction. (From Ref. 36.)... 

As light emission from aequorin is dependent on Ca2+, the protein has been widely employed for determination of this ion. In particular, the protein was used in the past in the highly sensitive measurement of intracellular calcium concentration in several kind of cells [16]. More recently immobilized aequorin was used to develop an optical biosensor for measurement of calcium ions in complex... 

June, C.H. and Rabinovitch, P.S., Flow cytometric measurement of intracellular ionized calcium in single cells with indo-1 and fluo-3, Methods Cell Biol., 33, 37, 1990. 

A final optical application deals with the measurement of intracellular nicotinamide adenine dinucleotide (NADH) by fluorescence [77], giving information about the physiological status of wastewater treatment plant biomass. This indirect method could be envisaged for toxicity estimation. 

The difficulties of intensity-based flow cytometry are illustrated by the present difficulties of cell-by-cell measurements of intracellular calcium. This can be accomplished using the calcium probe Indo-l,(34 38) but requires an ultraviolet (UV) laser source which is not routinely available in flow cytometry (Indo-1 is an emission wavelength ratiometric probe). Flow cytometers routinely have argon ion laser sources with outputs of 488 or 514 nm. Measurement of intracellular ions other than Ca2+ is nearly impossible. (The SNAFL and SNARF probes should allow pH measurement from the wavelength-ratiometric data.(15))... 

C.K. Rhee, L.A. Levy, R.E. London, Fluorinated o-aminophenol derivatives for measurement of intracellular pH, Bioconjug. Chem. 6 (1995) 77-81. 

E. Murphy, Measurement of intracellular ionized magnesium, Miner. Electrolyte Metab. 19 (1993) 250-258. 

Some other nuclei. Here are a few reported uses of NMR on other nuclei. 3He, binding into little cavities in fullerenes 478 nB, binding of boronic acids to active sites 479 23Na, measurement of intracellular [Na1] 180 482 35C1 and 37C1, binding to serum albumin 483 113Cd,... 

AP-induced toxicity and measurement of cell survival/injury were performed as described in detail elsewhere.1011 Briefly, 6-day-old cells were exposed to fresh solutions of either Ap25 35 or Ap, 42 for 24 h, in the presence or absence of different drugs. Cell survival and extent of cell death were determined using MTT and Sytox green assays, respectively. Measurement of intracellular reactive oxygen species was determined by dichlorofluorescein (DCF) fluorescence assay, as described previously.23... 

Slawson SE, Roman BB, Williams DS, Koretsky AP (1998) Cardiac MRI of the normal and hypertrophied mouse heart. Magnetic Resonance in Medicine 39 980-987 Szczepaniak LS, Babcock EE, Schick F et al. (1999) Measurement of intracellular triglyceride stores by H spectroscopy validation in vivo. American Journal of Physiology 276 E977-989... 

BERG W, HORNBECK P (1993) A micro-radioimmunoassay for the measurement of intracellular cAMP. BioTediniques, 15,56-59. 

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