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An agarose-based cloning-ring anchoring method for isolation of viable cell clones.

ABSTRACT: Isolation of clonal cell populations is a crucial aspect of cell biology during engineering of specific cell strains with both genotypic and phenotypic variations. The use of cloning rings is the most established method, but requires anchoring chemicals or material that can often interfere with quantitative clonal-cell isolation and causes physical damage to the cells. Here we report a non-toxic, cell culture-compatible method that uses aga-rose for embedding the cloning rings during isolation of cell clones from monolayer cultures, with enhanced cell-viability and reproducibility during downstream applications. The method is simple and rapid, minimizing the chances for desiccation or cross-contamination during colony-lifts.

SUBMITTER: Mathupala S

PROVIDER: S-EPMC2727865 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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An agarose-based cloning-ring anchoring method for isolation of viable cell clones.

Mathupala Saroj S Sloan Andrew A AA

BioTechniques 20090401 4

Isolation of clonal cell populations is a crucial aspect of cell biology during engineering of specific cell strains with both genotypic and phenotypic variations. The use of cloning rings is the most established method, but requires anchoring chemicals or material that can often interfere with quantitative clonal-cell isolation and causes physical damage to the cells. Here we report a non-toxic, cell culture-compatible method that uses aga-rose for embedding the cloning rings during isolation o ...[more]

PMID: 19450237

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Project description:MotivationB cells derive their antigen-specificity through the expression of Immunoglobulin (Ig) receptors on their surface. These receptors are initially generated stochastically by somatic re-arrangement of the DNA and further diversified following antigen-activation by a process of somatic hypermutation, which introduces mainly point substitutions into the receptor DNA at a high rate. Recent advances in next-generation sequencing have enabled large-scale profiling of the B cell Ig repertoire from blood and tissue samples. A key computational challenge in the analysis of these data is partitioning the sequences to identify descendants of a common B cell (i.e. a clone). Current methods group sequences using a fixed distance threshold, or a likelihood calculation that is computationally-intensive. Here, we propose a new method based on spectral clustering with an adaptive threshold to determine the local sequence neighborhood. Validation using simulated and experimental datasets demonstrates that this method has high sensitivity and specificity compared to a fixed threshold that is optimized for these measures. In addition, this method works on datasets where choosing an optimal fixed threshold is difficult and is more computationally efficient in all cases. The ability to quickly and accurately identify members of a clone from repertoire sequencing data will greatly improve downstream analyses. Clonally-related sequences cannot be treated independently in statistical models, and clonal partitions are used as the basis for the calculation of diversity metrics, lineage reconstruction and selection analysis. Thus, the spectral clustering-based method here represents an important contribution to repertoire analysis.Availability and implementationSource code for this method is freely available in the SCOPe (Spectral Clustering for clOne Partitioning) R package in the Immcantation framework: www.immcantation.org under the CC BY-SA 4.0 license.Supplementary informationSupplementary data are available at Bioinformatics online.

Project description:As an alternative to laser-based methods, we developed a novel in situ cell isolation method and instrument based on local water absorption of millimeter wave (MMW) radiation that occurs in cellular material and nearby culture medium while the cultureware materials (plastic and glass) are transparent to MMW frequencies. Unwanted cells within cell population are targeted with MMWs in order to kill them by overheating. The instrument rapidly (within 2-3 seconds) heats a cell culture area of about 500 µm in diameter to 50 °C using a low-power W-band (94 GHz) MMW source. Heated cells in the area detach from the substrate and can be removed by a media change leaving a bare spot. Hence we named the instrument "CellEraser". Quick, local and non-contact heating with sharp boundaries of the heated area allows elimination of the unwanted cells without affecting the neighboring cells. The instrument is implemented as a compact microscope attachment and the selective hyperthermic treatment can be done manually or in an automated mode. Mammalian cells heated even momentarily above 50 °C will not survive. This "temperature of no return" does not compromise cellular membranes nor does it denature proteins. Using the CellEraser instrument we found that the key event that determines the fate of a cell at elevated temperatures is whether or not the selectivity of its nucleus is compromised. If a cell nucleus becomes "leaky" allowing normally excluded (cytoplasmic) proteins in and normally nuclear-localized proteins out, that cell is destined to die. Quick heating by MMWs to higher temperatures (70 °C) denatures cellular proteins but the cells are not able to detach from the substrate - instead they undergo a phenomenon we called "thermofixation": such cells look similar to cells fixed with common chemical fixatives. They remain flat and are not washable from the substrate. Interestingly, their membranes become permeable to DNA dyes and even to antibodies. Thermofixation allows the use of western blot antibodies for immunofluorescence imaging.

Dataset Information

An agarose-based cloning-ring anchoring method for isolation of viable cell clones.

Publications

An agarose-based cloning-ring anchoring method for isolation of viable cell clones.

Similar Datasets

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