Labeled cells

The inhibition of Streptococcus mutans adherence to hydroxyapatite with combinations of alkyl phosphates and nonionic surfactants was tested. Seven alkyl phosphate derivatives and three nonionic surfactants were examined for their ability to inhibit the adherence of 3H-labeled cells of S. mutans to hydroxyapatite treated with buffer or parotid saliva. No compound by itself effectively hindered binding of bacteria to hydroxyapatite. A combination of certain of the alkyl phosphates, notably a disodium phosphate of 1-octadecanol, and nonionic surfactant at a 1 1 molar ratio gave a strong inhibition of S. mutans adherence. Treatment with this combination resulted in 98% reduction of adherence. Adsorption of the two types of surface-active agents alone and in combinations was studied using 14C-labeled agents. Electrophoretic measure-... 

Figure 10. Dose-response curves for 6-HCH stimulation of cytosolic Ca increases measured in Indo-l-labeled cells. Experimental conditions are as in Figure 9.

Figure 11. Effects of EGTA or treatment with islet activating protein (lAP or pertussis toxin) on the 6-HCH-induced Ca response detected in Indo-l-labeled cells. Cells were treated for 2 hours at 37 C with (lAP) or without (Control) 10 Jg/mL lAP, then labeled with Indo-1. Cells were washed and resuspended at 2 x 10 cells/mL buffer and stimulated with 6-HCH (solid trace). In some cases (dashed traces), stimulation was preceded by the addition of 5-mAf EGTA 10 s before stimulation. Other experimental conditions are as in Figure 9.

NOTE Retrogradely labeled cells were counted after a fluorescent dye was injected in... 

Figure 1.2 Regression analysis between numbers of C-Fos IR + GAD double-labeled neurons and the amount of sleep in the 2 h preceding sacrifice. The numbers of double-labeled cells were highly correlated with the percentage of preceding sleep in VLPO and both rostral and caudal MnPN. From Gong et al. (2004b).

Figure 1.6 (A) Changes in c-Fos IR in hypocretin/orexin(HCRT+)- containing neurons in response to BIC treatment as a function of distance from the microdialysis probe. The percentage of double-labeled cells was greatest closer to the probe (grids 1 and 2, compared with 3 and 4) and was greater with higher concentrations or exposure times. Contralateral cells were not affected. (B) Much lower percentages of melanin-concentrating hormone (MCH+) exhibited c-Fos IR activation in response to BIC. From Alam et al. (2005).

Fig. 3.11. FRET FLIM experiment to study colocalization of two lipid raft markers, GPI-GFP and CTB-Alexa594. The rows of images show intensity and lifetime images of donor-labeled and donor + acceptor-labeled cells. The histogram shows the lifetime distribution of the whole cells. The FRET...

We assembled a TIRFM with low magnification to study cell adhesion behavior on SAMs with various functional groups [42]. Figure lb shows a schematic illustration of the cell adhesion process and the corresponding TIRFM images. A suspension of cells with fluorescently labeled cell membranes is applied onto a substrate (Fig. lb-1). At first, no bright spots were observed by TIRFM,... 

The following protocol was used by Wilchek and Bayer (1987) to label cell-surface galactose residues. 

To label cell-surface azide-glycans, the cells are reacted for 1 hour using a final concentration of 1 mM biotin-PEG-phosphine reagent dissolved in PBS, pH 7.4. 

Rembaum, A., Margel, S., and Levy, J. (1978) Polyglutaraldehyde a new reagent for coupling proteins to microspheres and for labeling cell-surface receptors./. Immunol. Meth. 24, 239-250. 

Lymphocytes, the effector cells of the acquired immune system, include morphologically indistinguishable T and B cells, the former divided into CD4+ T helper cells and CD8+ cytotoxic T cells. Since the functions of those cell subsets differ so drastically, it became important to develop tools to distinguish them from each other. Efforts to identify cell subsets according to their expression of different surface antigens have been successful, including various Cluster of Determination (CD) markers (Table 23.1). In addition, cross-reactive monoclonal antibodies, and subsequently developed species-specific polyclonal and monoclonal antibodies towards the major histocompatibility complex (MHC) have been used to label cells in circulation and in tissue sections (Table 23.1). 

The stained nuclei stand out in vivid contrast to other fluorescently labeled cell structures when observed by fluorescence microscopy. 

This assay method (RIPA) is used primarily in research. It is too technically demanding for routine use in clinical laboratories. HIV is cultured in radio-labeled cells, or viral proteins are directly labeled with a radioisotope. The virus is disrupted and then exposed to the test specimen. Specific antigen-antibody complexes are concentrated and isolated by immunoprecipitation. After exten-... 

There have been rather few studies of the location of probes in whole cells. DPH incorporates into most subcellular fractions (see, e.g, Ref. 64), whereas with TMA-DPH, early after introduction only the plasma membranes appear to be labeled/64,65) There is considerable interest in examining the lipid motional properties of living cells by fluorescence techniques. In this type of study the location of the probe has to be carefully checked before conclusions can be drawn. This is carried out by separate measurements of the recovery of probe from intact labeled cells in isolated subcellular fractions and/or by fluorescence microscopy. 

Quantitative determination of the absolute distance from the surface to a labeled cell membrane at a cell/substrate contact region can be based on the variation of F(d) with 0.(1O6) This effort is challenging because corrections have to be made for 0-dependent reflection and transmission through four stratified layers (glass, culture medium, membrane, and cytoplasm), all with different refractive indices. For 3T3 cells, Lanni et a//1065 derived a plasma membrane/substrate spacing of 49 nm for focal contacts and 69 nm for close contacts elsewhere. They were also able to calculate an approximate refractive index for the cytoplasm of 1.358 to 1.374. 

A fluorescence-activated cell sorter (FACS) is a flow cytometry instrument used to separate and identify cells in a heterogeneous population. Cell mixtures to be sorted are first bound to fluorescent dyes such as fluorescein or phycoerythrin. The labeled cells are then pumped through the instrument and are excited by a laser beam. Cells that fluoresce are detected, and an electrostatic charge is applied. The charged cells are separated using voltage deflection. 

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