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1.
Langmuir ; 33(28): 6985-6990, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28666390

RESUMO

A method is described for the sensitive measurement of adsorbed proteins using femtoliter microwells. Quantitative measurement of adsorbed protein is demonstrated at surface densities from 10 fg/cm2 to 3 pg/cm2. Determination of the efficacy of barrier coatings is also demonstrated using femtoliter microwells. Adsorption at low surface densities is measured, indicating the highest affinity sites on the surface and therefore the initial stages of adsorption. The femtoliter microwell method is shown to be useful in detecting differences between effective protective coatings.


Assuntos
Adsorção , Propriedades de Superfície
2.
Methods Mol Biol ; 2377: 29-42, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709609

RESUMO

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells. However, most such screens have been performed in vitro with little consideration into how medium composition might affect gene essentiality. This protocol describes a method to use CRISPR/Cas9-based loss-of-function screens to ask how gene essentiality in human cell lines varies with medium composition. First, a single-guide RNA (sgRNA) library is packaged into lentivirus, and an optimal infection titer is determined for the target cells. Following selection, genomic DNA (gDNA) is extracted from an aliquot of the transduced cells. The remaining transduced cells are then screened in at least two distinct cell culture media. At the conclusion of the screening period, gDNA is collected from each cell population. Next, high-throughput sequencing is used to determine sgRNA barcode abundances from the initial and each of the final populations. Finally, an analytical pipeline is used to identify medium-essential candidate genes from these screen results.


Assuntos
Sistemas CRISPR-Cas , Genes Essenciais , Sistemas CRISPR-Cas/genética , Linhagem Celular , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Guia de Cinetoplastídeos/genética
3.
Mol Ther Methods Clin Dev ; 24: 380-393, 2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35284590

RESUMO

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables, including proliferation, differentiation, function, activation, and mitochondrial phenotypes. The extent of proliferation and function depended on the culture media rather than stimulation conditions. Moreover, the expanded T cell end products adapted their metabolism when switched to a different media formulation, as shown by glucose and glutamine uptake and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Expanded T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. Thus, ex vivo T cell expansion media have profound impacts on metabolism and function.

4.
Cell Metab ; 33(6): 1248-1263.e9, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33651980

RESUMO

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly reflect metabolite availability in human blood. Here, we performed CRISPR-based screens in traditional versus human plasma-like medium (HPLM). Sets of conditionally essential genes in human cancer cell lines span several cellular processes and vary with both natural cell-intrinsic diversity and the combination of basal and serum components that comprise typical media. Notably, we traced the causes for each of three conditional CRISPR phenotypes to the availability of metabolites uniquely defined in HPLM versus conventional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.


Assuntos
Sistemas CRISPR-Cas , Meios de Cultura , Linhagem Celular Tumoral , Humanos
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