Fixation cell suspensions

A number of techniques are available, including laser microprobe mass analysis (LAMMA) and various microprobes, that are capable of determining within tissues or cell preparations the cellular and subcellular distribution of lead in situ. While these techniques generally require varying levels of sample preparation (e.g., tissue fixation, cell suspensions, etc.), they provide powerful tools for probing different cellular compartments and processes involving lead. Thus, the site-specific distribution of lead can be used to evaluate local toxicity, which can be correlated with pathological alterations in tissues. 

Morgan JM. A protocol for preparing cell suspensions with formalin fixation and paraffin embedding which minimises the formation of cell aggregates. J Cell. Pathol. 2001 5 171-180. 

Proteins can be detected in tissue sections or cell cultures using similar immune detection systems. Use of an antibody to detect specific proteins in tissues is called immunohistochemistry, whereas detection of proteins in cell suspensions is called immunocyto-chemistry. Tissues can be prepared by fixation and embedding in paraffin wax, or by rapid freezing in a compound that inhibits ice formation in the tissue, so as to preserve cell morphology. Some antibodies do not work well with paraffin-embedded tissues, probably because the antibody cannot access the antigen properly (133). The most common labeling system used for detection of the bound antibody is an enzyme-coupled secondary antibody that produces a color reaction... 

Venous blood was collected from healthy donors. Platelet rich plasma (PRP) was obtained by centrifugation of the citrated blood at 110 g for 10 min at the room temperature. The platelet suspension was incubated at 37 C for 30 min before the mica adherence studies were performed to allow platelets to reach a resting condition. Mica-activated platelets were prepared by micropipetting of suspended platelets onto the freshly cleaved mica surface. A drop of 1.5% glytaraldehyde was placed on mica substrates for fixation of mica-activated platelets for 30 min. Activated platelets in suspension were fixed with 1.5% glutaraldehyde for 30 min after the addition in the cell suspension of ADP alone or ADP with H2O2. Then platelets were washed five times in HEPES saline buffer, dehydrated in a graded series of ethanol and air dried. 

Tuovinen, O.H. and Kelly, D.P., (1974 (a) (b) (c). Studies on the growth of Thiobacillus ferrooxidans. II. Toxicity of uranium to growing cultures and tolerance conferred by mutation, other metal cations and EDTA. Arch. Mikrobiol., 95 153—164. III. Influence of uranium, other metal ions and 2,4-dinitrophenol on ferrous iron oxidation and carbon dioxide fixation by cell suspensions, ibid, 95 165—180. IV. Influence of monovalent metal cations on ferrous iron oxidation and uranium toxicity in growing cultures, ibid, 98 167—174. 

Fixation of larvae, adult worms or intact nurse cells, suspensed in cold PBS, was done by adding cold x2 Ruvkun fixation buffer, diluted with 2% formaldehyde to xl final concentration. Frozen samples were stored at -80°C. After thawing. 

Fixation with ethanol can begin immediately after a suitable cell suspension pellet is available, e.g., 2x 106 cells per tube (see Note 3). 

After fixation the specimen has to be embedded in a matrix to preserve the cell-to-cell relationship. For cell suspensions such as E. coli a 2-10% gelatin solution is often used which also improves the sectioning characteristics. The embedded material is then cut into small 0.5-1 mm cubes which are infused with 2.3 M sucrose to prevent freeze damage and to give the specimen the plasticity which is required for smooth ultrathin sectioning. 

Store the cell suspension immediately at 4 °C in the dark. Acquire samples as soon as possible. If you need to wait longer than 1 h before the acquisition, a fixation procedure is required. 

By combining a two-phase flow simulator and the photosynthesis model, Sato et al. [92,93] explored the amount of carbon fixation and the growth curve of microalgae. A similar approach was applied to simulate a tubular recycled PBR for macro-algal cell suspension cultures [49]. This model predicts a critical ceU density at which photosynthetic biomass production switches from a rate-limited process to a CO2 delivery-limited process. Rate-limited growth proceeds only to this critical cell... 

Spectra of cells dried from formalin do not show the characteristic absorption associated with protein aggregates, indicating that formalin fixation does not induce protein denaturation. However, drying from formalin does result in the appearance of a series of sharp absorptions between 1000 and 1500 cm"l. These narrow absorption bands arise from formaldehyde that is retained in salt crystals in the film. These distinctive absorptions are not present if the cell suspension is washed with isotonic... 

There are a number of different cell fixation and process methods used in laboratories worldwide. Fixation time in tissue will reflect a commonly accepted fixation time such as that seen in pre-analytical guidelines published in the package inserts for commercially available kits. Regarding cell lines points to note are how soon are the cells fixed after harvesting, are the cells fixed in suspension, or when are they in a suspension matrix such as agarose. 

The direct labeling technique may be applied to either unfixed cells or fixed tissue, provided that the antigen is stable to fixation. Often this must be determined empirically (see Chapter 8). The following protocol presents the simplest approach direct labeling of a surface antigen on unfixed cells in suspension. 

Longer fixation times may be necessary to fix the suspension grown cells adequately to the plate. 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

SpA-containing Staphylococci (Section 3.3), fixed with trichloroacetic acid (TCA) or formalin (Section 3.3.1), may also serve as immunosorbent for many mammalian antibodies (Table 7.1). Both fixation procedures are satisfactory but yield products with different properties. Fixation of Staphylococci with hot TCA (Lindmark, 1982) removes the negatively charged cell-wall polymer teichoic acid, producing an IgG-sorbent which can bind 1.4 mg human IgG per ml of a 10% (v/v) suspension of bacteria and is stable for about 5 months. Formalin-fixed bacteria (Kessler, 1976) bind 35% more IgG and are stable for at least 1 year. However, IgG can be eluted quantitatively from TCA-fixed bacteria but not from formalin-fixed bacteria, probably due to the interaction between IgG and teichoic acid, unless 80 mM MgCh is included in the acid buffer. 

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