4 amino 5 methylamino 2 7 difluorofluorescein diacetate daf

4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF) (ThermoFisher) was used to measure and spatially resolve nitric oxide (NO) production in disks. 7 days after chamber placement, wells were washed three times in 1× PBS to remove all phenol red and BSA, which interfere with DAF fluorescence. To ensure that the lower compartment of the chamber was adequately washed, 1× PBS was gently pipetted up and down directly over the opening. The 4T1 cells were then incubated in phenol red-free, serum-free media with DAF (10 µM) and Hoechst (µg mL⁻¹) at 37 °C for 45 min. This dye was often combined with TMRE in order to obtain simultaneous measurements of mitochondrial membrane polarization state. Samples were then immediately imaged on a Zeiss 710 Laser Scanning Confocal Microscope with a ×10 dry objective using standard FITC emission and excitation filters.

NaCl, D-glucose, KH₂PO₄, NaF, dihydroethidium (DHE, stock 10 mM in DMSO), Tris-base, Dulbecco's Modified Eagle Media (DMEM), fetal calf serum (FCS), penicillin/streptomycin, 1x minimal essential amino acids, and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF, stock 10 mM in DMSO) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Acetylcholine, phenylephrine, leupeptin, aprotinin, sodium orthovanadate, pepstatin, phenylmethylsulfonyl fluoride (PMSF), NaHCO₃, ethylene glycol tetraacetic acid (EGTA), A23187 (calcium ionophore, stock 10 mM in DMSO), sodium deoxycholate, NP40, lucigenin, diethylenetriaminepentaacetic acid (DTPA), apocynin, and endothelial cell growth supplement were all purchased from Sigma-Aldrich (St. Louis, MO, USA). KCl, CaCl₂, MgSO₄, and ethylene-diaminetetraacetic acid (EDTA) were all purchased from EMD Millipore (Billerica, MA, USA). Sodium dodecyl sulfate (SDS) and NADPH were purchased from VWR (Radnor, PA, USA). Alexa-647-labeled Annexin V were purchased from BioLegend (San Diego, CA, USA). Heparin was purchased through Leo Pharma (Thornhill, ON, Canada). A complete list of all antibodies used and their sources is provided in Table 1.