title: A minimally invasive method for retrieving single adherent cells of different types from cultures.
abstract: 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.
authors: 1] Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501 [2].
authors: Jia
authors: J
authors: Zeng
authors: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501.
authors: Aida
authors: A
authors: Mohammadreza
authors: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501.
authors: Weimin
authors: W
authors: Gao
authors: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501.
authors: Saeed
authors: S
authors: Merza
authors: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501.
authors: Dean
authors: D
authors: Smith
authors: 1] Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501 [2].
authors: Laimonas
authors: L
authors: Kelbauskas
authors: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501.
authors: Deirdre R
authors: DR
authors: Meldrum
mesh_terms: D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods*; D013314:Stress, Mechanical
chemical_list: D008164:Luminescent Proteins