Dcf da kit

Dulbecco's modified Eagle's medium (DMEM), antibiotic antimycotic solution, hydrogen peroxide (H₂O₂), 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT), dimethyl sulfoxide (DMSO), and 2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH) were purchased from Sigma‐aldrich (St. louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco (Waltham, MA, USA). RIPA buffer was purchased from Thermo Fisher (Waltham, MA, USA). Phosphate inhibitor and protease inhibitor were purchased from Gen DEPOT (Barker, TX, USA). DCF‐DA kit was purchased from Abcam (Cambridge, UK), and caspases‐3 was from Cayman chemicals (Ann Arbor, MI, USA). Antibodies against caspase‐3, caspase‐6, and caspase‐7 were obtained from Cell signaling Technology (Danvers, MA, USA). Antibody against Nrf2 was from Thermo Fisher scientific. Antibodies against heme oxygenase (HO), Lamin B, and β‐actin were from Santa Cruz (Dallas, TX, USA). ECL prime was purchased from GE Healthcare life sciences (Buchinghamshire, UK).

The intracellular ROS generation was quantified by confocal microscopy using the DCFDA Kit from Abcam with light modifications. Cell culture Petri dishes coated with poly-d-lysine were used to seed 250,000 MCF-7 cells in DMEM media and incubated for 12 h at 37 °C in 5% CO₂. The cultivated cells were treated with P22CYP-PpIX-PEG-(EST) or P22CYP-PpIX (35 μg/mL) and further cultured for 24 h. The cells were incubated with DCFDA (20 μM) in DMSO for 30 min at 37 °C in darkness and then exposed to UV light at 365 nm (3 J/cm²). The UV dose was monitored with a UV radiometer (VLX-3 W, Vilber Lourmat). The treated cells were rinsed twice with PBS (1×), fixed with 4% formaldehyde in PBS at 4 °C for 15 min. After fixation, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min at 4 °C and counterstained DAPI (0.25 ng/mL), followed by eight washes with PBS. The intracellular ROS content in MCF-7 cells treated with the particles (35 μg/mL) was analyzed on the basis of green fluorescence of DCFDA by confocal microscopy at 485 nm excitation and fluorescent emission at 535 nm.

To assess ROS production induced by oxidative stress in the mixed glial cells, a DCFDA kit (Abcam) was used following manufacturer’s specifications. Glial cells were stained for 30 min with 20 µM DCFDA, and then tert-butyl hydroperoxide (TBHP) was added at a concentration of 5 µM to induce oxidative stress (Roy and Sil 2012 (link)) with/without the addition of pre- or post-expanded CD34 + cells placed in a transwell for 24 h. Fluorescence was measured using a microplate reader with an ex/em 485/535 nm.