8 well chamber slide

To measure the expression level of NF-κB in CM from HEKn cell cultures or PBS-treated CCD-986sk cells, the cells were seeded at 3 × 10⁵ cells/well in 8-well chamber slides (Nunc, Waltham, MA, USA) for 12 h. After applying CM from HEKn cell cultures or PBS for 24 h, the cells were washed with PBS. Then the cells were fixed with cold-4% PFA for 5 min and washed with PBS. The slides were treated with normal serum for 1 h at room temperature to reduce nonspecific antigen–antibody interactions. NF-κB primary antibodies were incubated (listed in Supplementary Table S1) for 24 h at 4 °C and then washed with PBS. Then the slides were loaded with secondary antibody (Alexa Fluor 488; Invitrogen, Waltham, MA, USA) for 1 h at room temperature and rinsed with PBS. After washing with PBS, the samples were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Burlington, MA, USA) solution for 30 s at room temperature to stain the nuclei, rinsed with PBS, and then mounted by using a vector shield solution (Vector Laboratories, Burlingame, CA, USA). The slides were imaged with a confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany), and the images were analyzed with Zen 2009 software (Carl Zeiss).

The cells (4×10³ cells/well) were seeded in a 96-well plate and treated with different concentrations of panobinostat for 24, 48 and 72 h. The cell viability was detected using Cell Counting Kit-8 (CCK; Dojindo, Kumamoto, Japan) and was measured at 450 nm using a micro-ELISA reader (Molecular Devices, Sunnyvale, CA). The percentage of cellular viability was averaged and normalized against the untreated control (DMSO). The half maximal inhibitory concentration (IC₅₀) values were estimated for further studies. The cells were seeded (1 × 10⁴/well) on 8-well chamber slides (Nunc, Rochester, NY) and fixed. For the proliferation analysis, the fixed cells were incubated with 5-ethynyl-2′-deoxyuridine (EdU) using the Click-it EdU assay kit (Invitrogen, Carlsbad, CA) at 48 h, according to the manufacturer's protocol. The results were analyzed by a confocal microscope (Carl Zeiss, Jena, Germany). EdU-positive cell number against the total cell number.

NET production was performed as previously described (Von Köckritz-Blickwede et al., 2010 (link)). Briefly, 2 × 10⁵ neutrophils in HBSS+/+ were seeded in a 96-wells plates, and 25 nM PMA (positive control) or B. anthracis at approximately an MOI of 5 were added to the wells. The plate was spun down at 290 × g for 5 min and incubated at 37°C, 5% CO₂ for 4 h. Next, 500 U/ml of micrococcal nuclease (Sigma) was added to the wells and incubated at room temperature to digest the NETs. Then 5 mM EDTA was added to stop the activity of the micrococcal nuclease and cells removed by centrifugation. Supernatant was collected and incubated with Quanti-iT PicoGreen for 5 min at room temperature. Fluorescence signal was measured at excitation 480 nm/emission 520 nm on a SpectraMax Gemini EM fluorescence reader. To visualize NET structures, 5 × 10⁴ neutrophils in HBSS+/+ were seeded in borosilicate 8-well chamber slides (Nunc). Next 25 nM PMA (positive control) or 25 × 10⁴ CFU of B. anthracis in HBSS+/+ were added to each chamber. Slides were then incubated at 37°C, 5% CO₂ for 4 h, after which 4% paraformaldehyde was added and the content of the chambers fixed for 30 min at room temperature. Chambers were then washed three times with PBS, and NET structures stained with 5 μM SYTOX Green (Invitrogen). NETs were imaged on a Zeiss Observer D1 inverted fluorescent microscope.