Facs canto 1 flow cytometer

The intracellular production of ROS after treatment of H. capsulatum (EH-315) with the MIC₉₀ concentrations of the compounds 7 (0.122 µg/mL), 11 (0.24 µg/mL), and 32 (3.90 µg/mL) was evaluated using 50 µM of H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate, Invitrogen- Thermo-Fisher-Scientific, Waltham, MA, USA). As a control, treatments with AmB (0.06 µg/mL), ITZ (0.125 µg/mL) and hydrogen peroxide (10 mM–H₂O₂) were used. The treatment was carried out as described in Section 2.5.1. After the incubation time (4 h), the samples were washed, and 500 µL of PBS was added, transferred to the cytometer tubes. Then, 1.5 µL of the H₂DCFDA solution was added, incubated at RT, and protected from light for 10 min [62 (link),63 (link),64 (link)]. The samples were analyzed on a BD FACS Canto I flow cytometer localized at the Laboratory of Proteomics/Clinical Mycology, Faculty of Pharmaceutical Sciences, UNESP-Araraquara, Brazil.

Cells plated in 6-well plates were treated with flavopiridol, carfilzomib, the combination, or vehicle for 24–60 hours, depending on the cell line. Cells were trypsinized and fixed for 30 minutes in 70% ethanol at 4°C and stained with 50 mg/mL of propidium iodide containing 100 mg/mL of ribonuclease A. Flow cytometry was performed on a BD FACSCanto I flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences, San Jose, CA). Data were generated for at least 20,000 events per sample and analyzed using FlowJo^® version 10.2 software (FlowJo, LLC, Ashland, OR).

Intracellular ROS production after treating T. rubrum ATCC 28189 with 2-chalcone in the dark and 2-chalcone mediated PDT was evaluated using 50 µM H2DCFDA (2.7 dichlorodihydrofluorescein diacetate, Invitrogen) as described by Singulani et al. (2019) (link). This compound is converted to a highly fluorescent 2ʹ, 7ʹ-dichlorofluorescein (DCF) compound after cleavage of its acetate group by intracellular esterases and this compound binds to ROS. As controls, AMB and FLZ treatments were used. The treatment was performed as described apoptosis/necrosis assay section. After the incubation period, the samples were washed, following suspension in 500 µL of PBS, and transferred to cytometer tubes. Then, 1.5 µL of the H2DCFDA solution was added with subsequent incubation at 25°C in the dark for 10 minutes. The samples were analyzed on a BD FACS Canto I flow cytometer.