|Spectrophotometry, fluorometry and chemiluminescence
Spectrophotometric methods are frequently used because of their widespread application in quantitation of both kinetic or endpoint data. Spectrophotometric methods are generally more cost-effective, but lack the dynamic range and sensitivity obtained with fluorescence or chemiluminescence techniques. It is for this reason that many techniques require fluorescence or chemiluminescence to detect the low abundance of some biomolecules or enzymes during experimentation. These techniques are fundamental in our laboratory and have found many uses in both cell-free and cell-based studies investigating mechanisms of action and/or toxicity. Assays that have been optimised for our laboratory include determination of protein content, oxidative stress, cytochrome P450 activity, mitochondrial homeostasis, steatosis, glucose uptake, insulin secretion, modes of cell death and cytotoxicity. The department houses two fluorescence microplate readers, each capable of spectrophotometric (λ: 200 nm - 800 nm), fluorescent (λ: 300 nm - 700 nm) and chemiluminescent measurements. Possible advanced techniques include fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), fluorescence anisotropy (polarization), time-resolved fluorescence (TRF), homogeneous time resolved fluorescence (HTRF), time-resolved fluorescence resonance energy transfer (TR-FRET), Alphascreen® and Lanthascreen®.
Flow cytometric techniques are generally used to investigate changes in intracellular signalling, organelle homeostasis, membrane receptor expression and mode of cell death. These methods are more qualitative in nature and are useful in detecting changes, but can be used to quantify the relative degree of change. They have widespread use in classifying types of cells using the cluster of differentiation (CD) surface antigens, which allows for examining (or even isolating) very specific population of cells, such as different types of leukocytes and stem cells. Flow cytometry that have been utsed in our laboratory in the past include those to examine oxidative stress, mitochondrial homeostasis, steatosis, mode of cell death and cytokine expression profiling. The department houses a Beckman-Coulter FC500 with a 32-tube carousel-type autosampler. It is equipped with argon and xenon lasers for excitation and is capable of fluorescence emission measurement at λ: 525 nm (FL-1), 575 nm (FL-2), 620 nm (FL-3), 650 nm (FL-4) and 675 nm (FL-5).
Microbial infections are still a problem to date, with new resistant strains of bacteria appearing on a continuous basis. Due to this the development of novel antimicrobial agents is a definite must. Furthermore, the importance of biofilms in microbial disease is becoming more apparent. Through the use of screening analyses, such as the disc diffusion, broth micro-dilution, checkerboard, resazurin, MBEC and microscopic assays, one can determine the potential of compounds to elicit an antimicrobial effect. Various microbial strains and clinical strains are present within the department’s dedicated microbiology laboratory, including Staphylococcus, Escherichia, non-pathogenic Mycobacterium and Candida, to name a few.
The department has advanced cell culture facilities where a battery of cancer cell lines are maintained in culture and used to assay cytotoxic or cytostatic effects of various compounds of natural or synthetic origin. As cancer remains one of the most prevalent age related diseases, it is important to screen for potential beneficial anti-cancer agents. Various cytotoxicity and endpoint assays are implemented to assess the efficacy of agents, including cell survival studies, cell enumeration studies, mode of death studies, cell cycle analyses and assessing reversal of drug-resistance. Cancer cell lines available in our laboratory include breast carcinoma, cervical carcinoma, colon carcinoma, hepatoma, leukaemia and neuroblastoma. Methods to establish a number of primary cell cultures as well as isolated lymphocytes and neutrophils are available to serve as healthy controls for the determination of tumour specificity.
|Specialised quantitiatve analysis using liquid chromatography-tandem mass spectrometry
Through the use of high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS-MS), compounds can accurately be identified and quantified within complex mixtures and matrixes. It is often used to determine whether active ingredients meet specified limits within commercial products. Apart from this, it also allows researchers to quantitatively describe the fate of therapeutic or toxic compounds after being introduced into animals or humans, as well as profiling the products of metabolism of such compounds. Our laboratory currently has three HPLC systems that can be used in conjunction with a number of detection methods, including fluorescence detection, UV-Vis detection and MS-MS. Supporting infrastructure includes equipment for homogenisation, manual and automated SPE extraction and other methods of sample preparation.