Table 1.

Troubleshooting with the DNA-based tension probe system.

Step	Problem	Possible reason	Solution
2.7	Surfaces are not pink after AuNP incubation	Insufficient etching	Include a base-etching step after piranha etching. Etch coverslips in 0.5 M KOH and etch in 0.5M KOH in ice-bath with sonication.
		APTES is hydrolyzed	Use new APTES
		NHS reagents are hydrolyzed	Use new PEG-NHS reagents
		PEG-NHS reagents are hydrolyzed before being added to the surface	Work faster
		AuNP aggregated	Use fresh AuNP
		Residue water on the coverslips diluted the AuNP	Make sure there is no/little water residue before adding AuNP
3.3.2	Cells don’t spread on the surface	Low DNA probe density	If the surfaces are not as pink, troubleshoot as described in discussion; If the AuNP functionalization is normal, synthesize and use new DNA
		Low ligand density	Use fresh streptavidin and ligand reagents
		Cells donť spread on the ligand	Check cell spreading on a ligand-coated surface, if the cells donť spread still, include adhering molecules as described in ref12.
	Cells spread well but no tension signal is observed	The receptor-ligand interaction does not have force transmission	NA
		Force is short-lived or force signals are sparse	Use locking oligonucleotide
3.3.3	No locking signal is observed in Atto647N	The oligonucleotide has degraded	Check with MALDI-TOF-MS
	Locking signal is antilocalized with real-time probe	The receptor force is higher than the working range of locking nucleotide	Use 19 pN real-time hairpin probe from ref. 12 as described.