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91.
Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative
characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability
to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown
on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we
compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show
that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to
methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen
and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required
which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done
immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across
a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale
clonogenic experiments.
相似文献
Kishore GudaEmail: |
92.
Coral microbial communities, zooxanthellae and mucus along gradients of seawater depth and coastal pollution 总被引:3,自引:0,他引:3
The high incidence of coral disease in shallow coastal marine environments suggests seawater depth and coastal pollution have an impact on the microbial communities inhabiting healthy coral tissues. A study was undertaken to determine how bacterial communities inhabiting tissues of the coral Montastraea annularis change at 5 m, 10 m and 20 m water depth in varying proximity to the urban centre and seaport of Willemstad, Cura?ao, Netherlands Antilles. Analyses of terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA gene sequences show significant differences in bacterial communities of polluted and control localities only at the shallowest seawater depth. Furthermore, distinct differences in bacterial communities were found with increasing water depth. Comparisons of TRFLP peaks with sequenced clone libraries indicate the black band disease cyanobacterium clone CD1C11 is common and most abundant on healthy corals in less than 10 m water depth. Similarly, sequences belonging to a previously unrecognized group of likely phototrophic bacteria, herein referred to as CAB-I, were also more common in shallow water. To assess the influence of environmental and physiologic factors on bacterial community structure, canonical correspondence analysis was performed using explanatory variables associated with: (i) light availability; (ii) seawater pollution; (iii) coral mucus composition; (iv) the community structure of symbiotic algae; and (v) the photosynthetic activity of symbiotic algae. Eleven per cent of the variation in bacterial communities was accounted for by covariation with these variables; the most important being photosynthetically active radiation (sunlight) and the coral uptake of sewage-derived compounds as recorded by the delta(15)N of coral tissue. 相似文献
93.
Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane 总被引:2,自引:2,他引:0
Burtey A Rappoport JZ Bouchet J Basmaciogullari S Guatelli J Simon SM Benichou S Benmerah A 《Traffic (Copenhagen, Denmark)》2007,8(1):61-76
The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells. 相似文献
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96.
We used high-fidelity PCR to amplify 2 overlapping regions of the ribosomal gene complex from the rodent fur mite Myobia musculi. The amplicons encompassed a large portion of the mite's ribosomal gene complex spanning 3128 nucleotides containing the entire 18S rRNA, internal transcribed spacer (ITS) 1,5.8S rRNA, ITS2, and a portion of the 5'-end of the 28S rRNA. M. musculi's 179-nucleotide 5.8S rRNA nucleotide sequence was not conserved, so this region was identified by conservation of rRNA secondary structure. Maximum likelihood and Bayesian inference phylogenetic analyses were performed by using multiple sequence alignment consisting of 1524 nucleotides of M. musculi 18S rRNA and homologous sequences from 42 prostigmatid mites and the tick Dermacentor andersoni. The phylograms produced by both methods were in agreement regarding terminal, secondary, and some tertiary phylogenetic relationships among mites. Bayesian inference discriminated most infraordinal relationships between Eleutherengona and Parasitengona mites in the suborder Anystina. Basal relationships between suborders Anystina and Eupodina historically determined by comparing differences in anatomic characteristics were less well-supported by our molecular analysis. Our results recapitulated similar 18S rRNA sequence analyses recently reported. Our study supports M. musculi as belonging to the suborder Anystina, infraorder Eleutherenona, and superfamily Cheyletoidea. 相似文献
97.
Kais Z Barsky SH Mathsyaraja H Zha A Ransburgh DJ He G Pilarski RT Shapiro CL Huang K Parvin JD 《Molecular cancer research : MCR》2011,9(8):1091-1099
To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control. 相似文献
98.
99.
Background
Avida is a computer program that performs evolution experiments with digital organisms. Previous work has used the program to study the evolutionary origin of complex features, namely logic operations, but has consistently used extremely large mutational fitness effects. The present study uses Avida to better understand the role of low-impact mutations in evolution. 相似文献100.
Abedi-Ardekani B Kamangar F Sotoudeh M Villar S Islami F Aghcheli K Nasrollahzadeh D Taghavi N Dawsey SM Abnet CC Hewitt SM Fahimi S Saidi F Brennan P Boffetta P Malekzadeh R Hainaut P 《PloS one》2011,6(12):e29488