A systematic investigation of differential effects of cell culture substrates on the extent of artifacts in single-molecule tracking.
Single-molecule techniques are being increasingly applied to biomedical investigation, notwithstanding the numerous challenges they pose in terms of signal-to-noise ratio issues. Non-specific binding of probes to glass substrates, in particular, can produce experimental artifacts due to spurious molecules on glass, which can be particularly deleterious in live-cell tracking experiments. In order to resolve the issue of non-specific probe binding to substrates, we performed systematic testing of a range of available surface coatings, using three different proteins, and then extended our assessment to the ability of these coatings to foster cell growth and retain non-adhesive properties. Linear PEG, a passivating agent commonly used both in immobilized-molecule single-molecule techniques and in tissue engineering, is able to both successfully repel non-specific adhesion of fluorescent probes and to foster cell growth when functionalized with appropriate adhesive peptides. Linear PEG treatment results in a significant reduction of tracking artifacts in EGFR tracking with Affibody ligands on a cell line expressing EGFR-eGFP. The findings reported herein could be beneficial to a large number of experimental situations where single-molecule or single-particle precision is required.
Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, United Kingdom.
Laura C
LC
Zanetti-Domingues
Marisa L
ML
Martin-Fernandez
Sarah R
SR
Needham
Daniel J
DJ
Rolfe
David T
DT
Clarke
D000818:Animals; D016477:Artifacts; D016466:CHO Cells; D002417:Cattle; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002460:Cell Line; D003094:Collagen / Q000737:chemistry; D006224:Cricetinae; D005353:Fibronectins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000737:chemistry; D008024:Ligands; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013499:Surface Properties
D005353:Fibronectins; D005456:Fluorescent Dyes; D007797:Laminin; D008024:Ligands; D010455:Peptides; D049452:Green Fluorescent Proteins; D011107:Polylysine; D011092:Polyethylene Glycols; D003094:Collagen