Secretion from single cells

The three examples shown In this paper Indicate that mlcrovoltam-metrlc electrodes are useful tools to probe chemical heterogeneities In solution, and furthermore to characterize these phenomena under dynamic conditions. To achieve greater spatial resolution, smaller electrodes will need to be employed. Automation of this type of measurement would be desirable and can readily be accomplished with piezoelectric mlcroposItloners and other such devices which can be remotely controlled. Such developments will lead to a form of dynamic chemical microscopy which would be useful to measure such events as secretion from single cells, corrosion processes In pits and cracks, or further studies of solution flow. 

Allogeneic mixed thymocyte medium (4) is a conditioned medium used to aid cells recover from the fusion process. It also encourages division of hybridoma cells after dilution cloning when colonies derived from single cells are required. Rat thymi are used for this purpose to minimise the number of animals required to produce adequate numbers of cells. The medium contains a number of helper cytokines produced by the thymocytes. It is important that the thymocytes used are derived from two different rat strains as this causes co-stimulation and enhanced cytokine secretion by the cells. Mixed thymocyte medium must always be diluted with RPMI 1640 medium containing 15% FBS and is usually used at a dilution of 10-15%. 

Such electrodes have been used to examine insulin secretion from single pancreatic P cells. A stimulant is introduced to contact a single P cell adhering to the bottom of a petri dish. The microelectrode is brought into contact with the cell. The result (representing insulin secretion) is shown in Fig 14.45. The peaks shown are Ca2+ dependent, and this is characteristic of an exocytotic process (Section 14.10.1). The area under the peak represents 360,000 insulin molecules. The results show that the spikes correspond to the ejection of packets of insulin secreted in exocytosis. 

Plants Photosynthetic so don t need much feeding can be cloned from single cells products can be secreted from roots or in sap. Cell walls difficult to penetrate by vector slow growing multi-cellular. 

In contrast, constant potential amperometry has allowed the quantitative aspects of single exocytotic release events to be studied in detail. This technique provides specific information on the amplitude, kinetics, and location of individual release events from single cells. Secretion is resolved as a series of current spikes that represent the electrooxidation of released substances. Wightman et al. have shown that each amperometric current spike detected represents the oxidation of neurotransmitter from a single exocytotic event [8]. In addition, the technique holds the potential to provide clues about the fusion pore complex, which manifests itself as a pre-spike foot that is observed directly prior to some release events. A drawback of this technique, however, is that chemical identification must be sacrificed for temporal resolution. This is a concern when one considers the complex biological matrix present in synaptic vesicles. 

Qian, W. J. Aspinwall, C. A. Battiste, M. A. Kennedy, R. T. Detection of secretion from single pancreatic (3-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy. Anal Chem. 2000, 72, 711-717. 

A single cell suspension of LNC is prepared under aseptic conditions and cultured for various periods of time. The production by LNC of type lcytokines (such as IFN-y and IL-12) and type 2 cytokines (such as IL-4, IL-5, IL-10 and IL-13) is measured using cytokine specific ELIS As or cytokine microarrays. It is possible to measure the spontaneous production of IL-5, IL-10, IL-12, IL-13 and IFN-y by LNC without restimulation in vitro. However, IL-4 appears to be produced in comparatively small amounts and to induce detectable levels of this cytokine an additional stimulus is required concanavalin A (con A), a T lymphocyte mitogen. Culture of LNC derived from mice treated with chemical respiratory sensitizer with con A will stimulate the production of detectable levels of IL-4. Treatment with the same mitogen of LNC derived from naive or vehicle-treated mice fails to induce measurable IL-4 secretion [86],... 

---