A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures
| Autor: | Laimonas Kelbauskas, Deirdre R. Meldrum, Merza Saeed A, Dean Smith, Weimin Gao, Aida Mohammadreza, Jia Zeng |
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| Rok vydání: | 2014 |
| Předmět: |
Shear force
Cell Culture Techniques Gene Expression 02 engineering and technology Biology Bioinformatics Article 03 medical and health sciences Single-cell analysis Gene expression Cell Adhesion Humans Cell adhesion Gene Cells Cultured 030304 developmental biology 0303 health sciences Microscopy Confocal Multidisciplinary Reverse Transcriptase Polymerase Chain Reaction Reproducibility of Results 021001 nanoscience & nanotechnology Cell biology Trypsinization Luminescent Proteins Cell culture Stress Mechanical Single-Cell Analysis 0210 nano-technology Shear flow |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. |
| Databáze: | OpenAIRE |
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