4 amino 5 methylamino 2 7 difluorofluorescein daf fm diacetate

Acetaminophen (APAP; 4-acetamidophenol), Percoll, Hepes, (heparin sodium salt grade I-A from porcine intestinal mucosa), penicillin G (sodium salt), RPMI-1640 modified media (with L-glutamine and without sodium bicarbonate and phenol red), 0.4% trypan blue solution, GSH, GSSG, S-nitrosoglutathione (GSNO), 3NT, NADH, NAD, and trifluoperazine (TFP; 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine), ATP Bioluminescent Assay kit, were products of Sigma Chemical Company (St Louis, MO). Collagenase A from Clostridium histolyticum was purchased from Roche Diagnostics (Indianapolis, IN). MitoSOX Red, 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM), and JC1 were obtained from Life technologies (Eugene, OR). LDH (lactate dehydrogenase) cytotoxicity detection kit was a product of from Roche Diagnostic Corporation (Indianapolis).

A previously published protocol (Sapp et al. 2014 (link)) was followed to measure relative intracellular NO levels using DAF-FM diacetate staining. In brief, overnight cultures were diluted to an OD₆₀₀=0.05 in fresh biofilm medium, and 1 ml aliquots were used to inoculate wells of a 24 well plate (Costar 3524). Static biofilms were grown for 7 hours, followed by harvesting of the whole well (biofilm + planktonic cells) and resuspension in 1x Hank's Buffered Salt Solution (HBSS) containing 5μM 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM diacetate, Invitrogen Life Technologies). Cell suspensions were incubated at 37°C for 1 hour, followed by centrifugation and washing with 1 mL of HBSS. Washed cells were then either resuspended in 650μL of HBSS alone or in HBSS containing 100 μM DEA/NO (chemical NO donor, half-life = 2 min at 37°C, Cayman Chemicals). Samples were immediately loaded into a 96 well plate (Costar 3904) and the fluorescence (EX/EM 485±10/516±10) (RFU) and OD₆₀₀ readings of each sample were measured with a Biotek Synergy HT fluorescent microplate reader. Fluorescence (RFU/OD₆₀₀) was reported as relative fluorescence units (RFU) measured at 30 min divided by the OD₆₀₀ reading of each well. Relative fold-change in DAF-FM fluorescence for each experiment was calculated by dividing each RFU/OD₆₀₀ value by the average RFU/OD₆₀₀ of the wild-type strain.