Tetramethylrhodamine isothiocyanate (TRITC)

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.caut770
Keyword(s):  
Tetramethylrhodamine Isothiocyanate

scholarly journals ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS

1971 ◽  
Vol 49 (2) ◽  
pp. 390-404 ◽  
Author(s):  
George M. Maniatis ◽  
Vernon M. Ingram
Keyword(s):  
Experimental Error ◽  
Single Cells ◽  
Deae Cellulose ◽  
Fluorescein Isothiocyanate ◽  
Erythroid Cells ◽  
Fluorescent Staining ◽  
Ammonium Sulfate Precipitation ◽  
Blood Smears ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Cellulose Column

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.

scholarly journals Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes.

1981 ◽  
Vol 153 (6) ◽  
pp. 1615-1628 ◽  
Author(s):  
D T Fearon ◽  
I Kaneko ◽  
G G Thomson
Keyword(s):  
Cell Surface ◽  
Polymorphonuclear Leukocytes ◽  
Plasma Membranes ◽  
Antibody Fragment ◽  
Cell Receptor ◽  
Molar Ratio ◽  
Surface Receptors ◽  
C3b Receptor ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Human Polymorphonuclear Leukocytes

C3b receptors on human polymorphonuclear leukocytes (PMN) were nonrandomly distributed in small clusters on the plasma membranes of these cells when assessed by indirect immunofluorescence at 0 degree C using monospecific rabbit Fab' or F(ab')2 anti-C3b receptor and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat IgG anti-F(ab')2. When PMN were incubated with the bivalent anti-C3b receptor at 37 rather than at 0 degree C, almost no immunofluorescence was observed, which indicates that the C3b receptor-F(ab')2 complexes had been rendered inaccessible to TRITC-IgG anti-F(ab')2. Endocytosis of the anti-C3b receptor ligand was quantitated by measuring the binding 131I-IgG anti-F(ab')2 by PMN that had previously taken up 125I-F(ab')2 anti-C3b receptor at 0 and at 37 degree C, respectively. There was a constant 2: 1 molar ratio of anti-F(ab')2 to anti-C3b receptor with PMN that had been incubated with the first antibody at 0 degree C. In contrast, when increments of F(ab')2 anti-C3b receptor were taken up by the cells at 37 degree C, there was a dose-related decline in this molar ratio to a minimum of 0.2 molecules of anti-F(ab')2 anti-F(ab')2 bound per molecule of PMN-associated anti-C3b receptor. 125I-F(ab')2 anti-C3b receptor taken up by PMN at 37 degree C was also inaccessible to release by proteolytic treatment of the cells with pronase. The rate of endocytosis of 125I-F(ab')2 anti-C3b receptor was rapid as the PMN-bound antibody fragment became inaccessible to 131I-IgG anti-F(ab')2 within 10 min during incubation of the cells at 37 degree C. In contrast to these findings, 125I-Fab' anti-C3b receptor that was taken up by PMN at 37 degree C remained accessible to both 131I-IgG anti-F(ab')2 and to proteolytic release by pronase, which suggests that monovalent interaction of ligand with C3b receptors was not sufficient for induction of endocytosis. The requirement for multivalency was also demonstrated using the C3b-OR, the normal ligand for the C3b receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell surface, as determined by its accessibility to pronase, unless it was cross-linked with F(ab')2 anti-C3. Although C3b receptors on PMN do not mediate internalization of adsorptive pinocytosis of soluble ligand indicates their potential for the clearance of C3b-bearing immune complexes without recruitment of other cell surface receptors.

scholarly journals In vivo biodistribution of carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles in rats

2011 ◽  
Vol 26 (6) ◽  
pp. 619-627 ◽  
Author(s):  
V.H. Pereira ◽  
A.J. Salgado ◽  
J.M. Oliveira ◽  
S.R. Cerqueira ◽  
A.M. Frias ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fluorescein Isothiocyanate ◽  
Pamam Dendrimer ◽  
Brain Parenchyma ◽  
Morphological Alterations ◽  
Intracellular Drug Delivery ◽  
In Vivo Biodistribution ◽  
Tetramethylrhodamine Isothiocyanate ◽  
The Brain

Carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles, comprised of a PAMAM dendrimer core grafted with chains of CMCht, have recently been proposed for intracellular drug delivery. In previous reports, these nanoparticles had lower levels of cytotoxicity when compared with traditional dendrimers. In this study, the short-term in vivo biodistribution of fluorescein isothiocyanate (FITC)-labeled CMCht/PAMAM dendrimer nanoparticles after intravenous (IV) injections in Wistar Han rats was determined. The brain, liver, kidney, and lung were collected at 24, 48, and 72 h after injection and stained with phalloidin–tetramethylrhodamine isothiocyanate (TRITC, red) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) to trace the nanoparticles within these tissues. The liver, kidney, and lung were also stained for hematoxylin and eosin to assess any morphological alterations of these organs. CMCht/PAMAM dendrimer nanoparticles were observed within the vascular space and parenchyma of liver, kidney, and lung and in the choroid plexus, after each injection period. No particles were observed in the brain parenchyma, nor any apparent deleterious histological changes were observed within these organs. The CMCht/PAMAM dendrimer nanoparticles were stable in circulation for a period of up to 72 h, targeting the main organs/systems through internalization by the cells present in their parenchyma. These results provide positive indicators to their potential use in the future as intracellular drug delivery systems.

Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides

1985 ◽  
Vol 5 (6) ◽  
pp. 1229-1237
Author(s):  
W J Welch ◽  
J R Feramisco
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Stress Protein ◽  
Exchange Chromatography ◽  
Native Form ◽  
Step Procedure ◽  
Rapid Purification ◽  
Stress Dependent ◽  
Tetramethylrhodamine Isothiocyanate

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.

scholarly journals Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

1985 ◽  
Vol 5 (6) ◽  
pp. 1229-1237 ◽  
Author(s):  
W J Welch ◽  
J R Feramisco
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Stress Protein ◽  
Exchange Chromatography ◽  
Native Form ◽  
Step Procedure ◽  
Rapid Purification ◽  
Stress Dependent ◽  
Tetramethylrhodamine Isothiocyanate

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.

scholarly journals Cytochemical hybridization with fluorochrome-labeled RNA. II. Applications.

1981 ◽  
Vol 29 (2) ◽  
pp. 238-246 ◽  
Author(s):  
J G Bauman ◽  
J Wiegant ◽  
P van Duijn
Keyword(s):  
Fluorescence Microscopy ◽  
Dna Sequences ◽  
Cross Hybridization ◽  
Hydrazine Derivative ◽  
Crithidia Luciliae ◽  
Labeled Rna ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Quantitative Fluorescence ◽  
Hybridization Reaction ◽  
Adenovirus 5

The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNAs complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNAs labeled both with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNAs were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNAs to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was thus demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB cells a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells, or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.

scholarly journals Transport of Bacterial Lipopolysaccharide to the Golgi Apparatus

1999 ◽  
Vol 190 (4) ◽  
pp. 523-534 ◽  
Author(s):  
Nathalie Thieblemont ◽  
Samuel D. Wright
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Hela Cells ◽  
Brefeldin A ◽  
Cellular Responses ◽  
Epithelial Cell Line ◽  
Cholera Toxin B Subunit ◽  
B Subunit ◽  
Cell Line Hela ◽  
Tetramethylrhodamine Isothiocyanate

Addition of lipopolysaccharide (LPS) to cells in the form of LPS–soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)–dextran, LysoTracker™ Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)–ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY–ceramide and TRITC (tetramethylrhodamine isothiocyanate)–labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS–sCD14 complexes in a CD14-dependent fashion: BODIPY–LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A–treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.

Distribution of actin filament lengths measured by fluorescence microscopy

1992 ◽  
Vol 262 (3) ◽  
pp. C569-C577 ◽  
Author(s):  
S. Burlacu ◽  
P. A. Janmey ◽  
J. Borejdo
Keyword(s):  
Sample Preparation ◽  
Correlation Length ◽  
Actin Filament ◽  
Actin Filaments ◽  
Average Length ◽  
Potassium Acetate ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Good Agreement ◽  
Millimolar Concentration

We analyzed the distribution of actin filament lengths by optical microscopy (OM). OM avoids possible alterations in the size or structure of actin filaments occurring during sample preparation for electron microscopy (EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were analyzed for both size distribution and flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4, 25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM beta-mercaptoethanol] filaments did not aggregate into bundles and remained stable at nanomolar concentrations for at least 1 h. At the same concentration, actin labeled directly with rhodamine (no phalloidin) formed unstable filaments whose average length decreased with time. The number average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns, the ratio (rho) of the weight average length to the number average length was 2.06, and the correlation length (1/lambda) was 8.33 microns. These parameters were in good agreement with the values determined by EM for filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150 column before polymerization did not have a significant effect on the distribution of lengths but made filaments more stiff (1/lambda = 12.5 microns). Millimolar concentration of ATP increased the correlation length, and gelsolin had the expected fragmenting effect on filaments. These results show that OM can be used as a fast and reliable method to analyze the distribution and flexibility of actin filaments and suggest that, in spite of extensive manipulation of actin filaments during sample preparation, EM is a valid tool for determination of size parameters of actin filaments.

The Structures of Tetramethylrhodamine Isothiocyanate Isomers: An N. M. R. Study

1968 ◽  
Vol 1 (1) ◽  
pp. 23-26 ◽  
Author(s):  
D. Kramer ◽  
H. Klapper ◽  
F. Miller
Keyword(s):  
Tetramethylrhodamine Isothiocyanate
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