Clumped cells

Although this gel microdroplet method is still new (even after 10 years) and its potential applications relatively untested, it has been described here because it can teach us certain general lessons. It serves to remind us that cytometry, despite its name, does not necessarily involve the flow analysis of cells particles of many sorts will do just as well. It is also of interest as a method that has, in fact, institutionalized the formation of clumped cells that most workers try so hard to avoid. In addition, it has provided us with a way to make a small cell into a larger (flow-friendly) particle. Finally, it has given us inspiration by exemplifying the way in which lateral thinking can extend the impact of flow cytometry in new directions. 

The first medium change is done 24 hr later when fibroblast-like transformants are noted, particularly among foci of clumped cells. The medium is changed daily until sufficient numbers of dishes are available to compare delays in medium changes. 

A laser ablation TOF mass spectrometer has been developed to iderrtily individual airborne micrometer-sized particles, comprising a single cell or a small mrmber of clumped cells (Tobias et al. 2005). This approach is reagerrt-less, and it relies on laser ablation and detection of lower mass (less than m/z 200) positive and negative ions. MS signatrrres for aerosolized Mycobacterium tuberculosis particles are distinct fromM smegmatis. Bacillus atrophaeus, and B. cereus particles. This technique is tested as a stand-alone airborne M. tuberculosis detector in bioaerosols from an infected patient at airborne concentrations of 1 particle/liter. 

The direct microscopic count determines the number of viable and dead microorganisms ia a milk sample. A small amount (0.01 mL) of milk is spread over a 1.0 cm area on a microscope sHde and allowed to dry. After staining with an appropriate dye, usually methylene blue, the sHde is examined with the aid of a microscope (oil immersion lens). The number of bacterial cells and clumps of cells per microscopic field is determined and, by appropriate calculations, is expressed as the number of organisms per milliliter of sample. 

It has been known for more than a century that human blood can be classified into four blood-group types (A, B, AB, and 0) and that blood from a donor of one type can t be transfused into a recipient with another type unless the two types are compatible (Table 25.1). Should an incompatible mix be made, the red blood cells clump together, or agglutinate. 

A factor known as scatter factor has been characterized which causes the break up and stimulates motility of epithelial cell clumps (Stoker et al., 1987). This factor is identical to hepatocyte growth factor and increases the rate of locomotion of several other cell types. Motility factors elaborated from tumor cells are considered to play an important role in metastasis (see later). Guidance of cells by the physical topography of the substratum is another factor that profoundly affects the behavior of cells. 

Figure 4. Neurite outgrowth by LA-N-1 human neuroblastoma cells in culture. LA-N-1 human PNS neuroblastoma cells were grown for five days in N2 medium (as described by Bottenstein and Sato, 1979) on a polylysine and fibronectin-modified surface. The cells were plated in clumps, rather than as a single cell suspension, which enhances neurite extension. Very long processes result, and exhibit varicosities along their length. Most of the cells have migrated from the central clump. (Photo courtesy of Dr. jane Bottenstein.)...

Several carbohydrate-binding proteins have been isolated from Sambucus nigra and two haemagglutinins have been isolated from its bark. Its seeds contain a lectin that is related to the immunological properties of the bark. One of its carbohydrate-binding proteins, called nigrin, is made of two subunits and is reported to cause red blood cells to clump. 5... 

The source of free radicals is multiplied under these circumstances, arachidonic acid metabolism, activation of xanthine oxidase, perturbation of electron flow within the respiratory chain, and NOS activation. Structurally, excitotoxicity is generally described as a necrotic process involving initial swelling of the cell and of the endoplasmic reticulum, clumping of chromatin, followed by swelling of the... 

Initial classification of some cytokines was also undertaken on the basis of the specific biological activity by which the cytokine was first discovered (e.g. TNF exhibited cytotoxic effects on some cancer cell lines CSFs promoted the growth in vitro of various leukocytes in clumps or colonies). This, too, proved an unsatisfactory classification mechanism, as it was subsequently shown that most cytokines display a range of biological activities (e.g. the major biological function of TNF is believed to be as a regulator of both the immune and inflammatory response). More recently, primary sequence analysis of cytokines coupled to determination of secondary and tertiary structure reveal that most cytokines can be grouped into one of six families (Table 8.2). 

TNF-a can mediate death of sensitive cells via apoptosis or necrosis (necrotic death is characterized by clumping of the nuclear chromatin, cellular swelling, disintegration of intracellular organelles and cell lysis apoptotic death is characterized by cellular shrinking, formation of dense apoptotic masses and DNA fragmentation). 

The integrated system has been tested for enhancement of bacterial capturing by introducing a solution of bacteria spores (concentration of 106 cells per mL) at 200 pL min 1 flow rate into the flow cell. Here, a small number of spore clumps were observed forming in the central region of the chamber when the ultrasound (0.5 V at 1.92-MHz frequency) was turned on. The clumps were held stationary... 

This example treats a diffusion-reaction process in a spherical biocatalyst bead. The original problem stems from a model of oxygen diffusion and reaction in clumps of animal cells by Keller (1991), but the modelling method also applies to bioflocs and biofilms, which are subject to potential oxygen limitation. Of course, the modelling procedure can also be applied generally to problems in heterogeneous catalysis. 

Add 10 ml of trypsin solution (0.025% trypsin in PBS) to each flask. Once the cells have rounded up, the trypsin is neutralized by the addition of 10 ml of complete medium. A cell suspension is obtained by vigorous pipetting to break up cell clumps. 

Currently in phase II clinical trials as a treatment for melanoma, hepatocellular carcinoma, and non-small-cell lung cancer (Hamann 2004), the compound has a mode of action that is not well understood, though it has been deemed necrosislike and characterized by cytoplasmic swelling and DNA clumping (Janmaat et al. 2005). 

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