Cell staining procedures

Individual cells may be studied either by examining the cell morphology using a wide variety of specific cell-staining procedures and microscopy or by isolating individual cells and culturing them for further molecular studies. In order to examine cells on a subcellular or molecular level, it is necessary to extract and purify subcellular components such as DNA, RNA, proteins, and... 

Gram positive Bacterial cells which retain the crystal violet stain during a staining procedure. Most strains of bacilli are gram positive. 

The cell wall has a low affinity for dyes, which means that it is probably not stained in some of the usual staining procedures. It is lightly stained by certain basic dyes such as basic fuchsin and the methyl violets. Where deep staining of the wall is desired, the use of a mordant, such as tannic acid, is necessary. The mordant not only increases the affinity of the cell for dye, but it may increase the thickness of the wall. 

As in the preliminary experiment, root tips were collected after 5-7 days, subjected to the Feulgen staining procedure as described above, then prepared adequately as permanent slides, and finally subjected to microscope observation. For each treatment, fifteen root tips (5x3 replicates) were prepared and 30,000 cells (2,000 cells per root tip) were examined in the case of MH, and 150 metaphases (5x3 replicates) were examined in the case of COL. 

Gram Stain A staining procedure used in classifying bacteria. A bacterial smear on a slide is stained with a purple basic triphenyl methane dye, usually crystal violet, in the presence of iodine/potassium iodide. The cells are then rinsed with alcohol or other solvent, and then counter-stained, usually with safranin. The bacteria then appear purple or red according to their ability to keep the purple stain when rinsed with alcohol. This property is related to the composition of the bacterial cell wall. 

If the purpose of gel electrophoresis is to identify low-abundance proteins (e.g., low-copy-number proteins in a cell extract or contaminants in a purification scheme), then a high protein load (0.1 to 1 mg/ml) and a high-sensitivity stain such as silver or fluorescence should be used. When the intention is to obtain enough protein for use as an antigen or for sequence analysis, then a high protein load should be applied to the gel and the proteins visualized with a staining procedure that does not fix the proteins in the gel, e.g., colloidal CBB G-250 (Subsection 8.2.8.1). Furthermore, for purposes of quantitative comparisons, stains with broad linear ranges of detection response should be used. 

The following basic protocols may be used alone or combined with other staining procedures in multiparameter flow cytometry experiments. Although they are illustrated with data from cells that proliferate in suspension, these protocols may be easily modified for the analysis of cells isolated from tissues or adherent cells in culture, by incorporating an initial step for the preparation of single cell suspensions. The assays are conducted at room temperature, unless otherwise noted. 

Weak false-positive nuclear staining may occur with some monoclonal or polyclonal antibodies therefore, careful assessment of the reaction pattern with particular attention to the staining of the normal cells is recommended. False-positive nuclear staining may be accentuated if the slides are allowed to dry during the staining procedure. 

We believe that the /3 cell is a source of nitric oxide production by human islets because (1) IL-1 and IFN-induced nitric oxide production by human macrophages has not been clearly demonstrated (2) the cytokine combination of IL-1, IFN, and TNF induces the formation of nitric oxide by human islets either freshly isolated or cultured for 7 days at 25°C (a procedure which removes 80-90% of nonendocrine cells from the islet) and also by islets cryoperserved and (3) NADPH—diaphorase staining reveals that approximately 60-70% of human islet cells treated with cytokines stain for NADPH-diaphorase (J. A. Corbett and M. L. McDaniel, unpublished data). TTiis staining procedure has been shown to colocalize with nitric oxide synthase in a number of cells including rat islets (Corbett et al., 1993c), and nitric oxide synthase has been demonstrated to contain NADPH-diaphorase enzymatic activity (Dawson et al., 1991 Hope et... 

Prior to the staining procedures, fix cells in 70% ethanol. Best results are obtained by resuspending the cell pellet in 1 mL of PBS and adding 9 mL of ice-cold 70% ethanol while vortexing to prevent agglutination. Solid pieces of tissue can be fixed as 5 mm3 pieces directly in ice-cold 70% ethanol. Cells and tissues are usually left at least 1 h prior to staining. Fixed cells or tissues should be stored at 4°C and, m the author s experience, will remain suitable for BrdU staining studies for at least 3 yr. 

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

Differential staining technique Staining procedures that elicit differences between bacterial cells. 

Related Tests. Many cells exposed to test chemicals can be scored for chromosome aberrations by staining procedures followed by visual examination with the aid of the microscope. These include Chinese hamster ovary cells in culture treated in a protocol very similar to that used in the test for SCEs, bone marrow cells from animals treated in vivo, or lymphocytes from animals treated in vivo. The types of aberrations evaluated include chromatid gaps, breaks, and deletions chromosome gaps, breaks, and deletions chromosome fragments translocations and ploidy. 

Monocyte Monocytes are a class of white blood cells that co-purify with lymphocytes in commonly used density gradient procedures. They tend to be promiscuously sticky for the nonspecific (Fc) ends of monoclonal antibodies and therefore can lead to misleading results in analysis of leukocyte subpopulations unless their Fc receptors are blocked in the staining procedure. Monocytes differ from lymphocytes in their forward and side scatter characteristics. 

Figure 1. Polyphenotypic small round cell tumor from the abdomen of a 4-year-old boy. (a) The biopsied material stained weakly for CD99, whereas (b) the excised tumor showed diffuse and strong positivity for the same antigen. The staining procedure used was identical.

Figure 6. Staining obtained with two antibody clones to proliferating cell nuclear antigen (PCNA). Antigen retrieval and staining procedures were identical for the two antibodies, (a) Clone 19A2 showed a proliferating index of 75% compared to (b) to a 98% proliferating index in the adjacent section of breast cancer when stained with clone PC 10.

Ehrlich was born in Strehlen, Germany, and attended school in Breslau where an older cousin, Carl Weigert, was a physician at a local hospital. Weigert was researching cell staining with synthetic dyes, a procedure that makes cells more visible under a microscope. Weigert demonstrated the technique for his teenage cousin who was immediately fascinated by the process. 

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