Spectrostar nano mars v3

The extent of cell death was determined by a colorimetric method, based on the lactate dehydrogenase (LDH) enzyme activity in the supernatant, released from damaged cells [48 (link)]. Equal volumes (40 μL) of aspired media were mixed in a sterile 96-well plate with LDH reagent, containing 109 mM lactate, 3.3 mM ß-nicotinamide-adenine-dinucleotide-hydrate (#N7004), 2.2 U/mL diaphorase (#D2197), 3 mM TRIS, 30 mM HEPES, 10 mM NaCl, 350 μM thiazolyl blue tetrazolium bromide (all reagents were purchased from Merck), then incubated at 37 °C for 1 h. Absorbance was recorded at 570 nm and at 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Ortenberg, Germany).

To investigate the amount of fibrillar collagens, Sirius Red assay was performed (n = 5-well/treatment group). After 48 h of 30 v/v% PDE, 10 ng/mL TGF-β or 10 ng/mL PDGF-B treatment in the presence or absence of 4.5 × 10⁹–10¹⁰ particles/mL EVs P-MCs, PDE-FBs, or P-FBs were incubated in a fixative solution containing 26% ethanol, 3.7% formaldehyde, 2% glacial acetic acid for 15 min at room temperature. Samples were stained for 1 h at room temperature with a 0.1% solution of Sirius Red (DirectRed80) dissolved in 1% acetic acid, and then washed three times with 200 μL of 0.1 M HCl, and finally the bounded dye was dissolved by adding 100 μL of 0.1 M NaOH (all reagents were purchased from Merck, Germany). Absorbance was recorded at 544 nm and at 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Germany). Vehicle-treated (4 mM HCl in the case of TGF-β, 30% PDF the in case of PDE, and PBS in case of EV) cells served as controls. Results were normalized and determined as a percentage ratio of the control group values.

The extent of cell death was determined by a colorimetric method, based on the lactate dehydrogenase (LDH) enzyme activity in the supernatant, released from damaged cells [66 (link),67 (link)]. Equal volumes (40 μL) of aspired media were mixed in a sterile 96-well plate with LDH reagent, containing 109 mM lactate, 3.3 mM ß-nicotinamide-adenine-dinucleotide-hydrate (N7004), 2.2 U/mL diaphorase (D2197), 3 mM TRIS, 30 mM HEPES, 10 mM NaCl, 350 μM thiazolyl blue tetrazolium bromide (all reagents were purchased from Merck Life Science Kft. Budapest, Hungary), then incubated at 37 °C for 1 h. Absorbance was recorded at 570 nm and 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Ortenberg, Germany).