Phorbol 12 myristate 13 acetate pma

Splenic lymphocytes from mice were isolated according to a previously reported method [14 (link)], and these cells were stained with APC-conjugated anti-mouse CD25 and FITC-conjugated anti-mouse CD4 or isotypes (eBioscience, USA) for 20 min at 4 °C. These cells were washed twice, fixed, permeabilized and then stained with PE-conjugated anti-mouse Foxp3 for analysis of Treg subpopulations. To detect splenic Th17 cells, splenocytes were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (BioVision, Mountain View, CA, USA), 1 μg/ml ionomycin (Enzo Life Sciences, Farmingdale, NY, USA) and 500 ng/ml monensin (eBscience, San Diego, CA, USA) for 4 h and were stained with FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse IL-17 antibodies. Acquisitions were performed using a FACS Canto II flow cytometer (BD, USA). Data were analyzed based on the percentages of Th17 cells and Treg cells.

The formation of NETs was evaluated at basal state and at activated state using the membrane impermeable DNA-binding dye, SYTOX green (Molecular Probes, Invitrogen Life Technologies). Experiments were performed in 96-well culture plates. Freshly isolated neutrophils (2 × 10⁵/well) were stimulated with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA, 0.01 μg/mL final; Biovision, USA) for 1 h. Following PMA stimulation or basal state, cells were incubated with 2 μM SYTOX green to detect extracellular DNA. After 30 min, neutrophils were washed and resuspended in RPMI and the plates were read using the SpectraMAX Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) at excitation and emission wavelengths of 500 nm and 530 nm, respectively. The data were analyzed using SoftMax Pro Software (Molecular Devices). To measure ROS, isolated neutrophils were incubated at 37°C for 30 min in the presence of 2 μM 2′,7′-dichlorofluorescin diacetate (Molecular Probes), which is used to detect hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. To evaluate NET formation following 1 h PMA stimulation, extracellular fluorescence was measured using a spectrofluorometer at excitation and emission wavelengths of 488 nm and 527 nm, respectively.

Vorinostat was purchased from Sigma-Aldrich (Poole, UK) and panobinostat was ordered from Cambridge Bioscience (Cambridge, UK). They were each reconstituted in sterile DMSO (Sigma-Aldrich) to a concentration of 20 mM. Romidepsin was purchased from Abcam (Cambridge, UK) and reconstituted at a concentration of 2 mM in DMSO. Prostratin was obtained from Cambridge Bioscience.
Phorbol 12-myristate 13-acetate (PMA) was purchased from Abcam and reconstituted with DMSO at a concentration of 1 mg/mL. Recombinant interleukin-1β (IL-1β) was purchased from Life Technologies (Paisley, UK) and reconstituted at a concentration of 1 μg/mL in pure dH₂O.