Mmp 12

All reagents and acetazolamide,^{[16 (link)]} were obtained from Sigma-Aldrich. 1,2-HOPO-2,^{[25 (link)]} CGS^{[12 (link)]} and SAHA^{[26 ]} were prepared by literature methods. Absorbance assays were performed using a BioTek Synergy HT microplatereader. Fluorescence assays were performed using either a BioTek FLx800 microplate reader or a BioTek Synergy HT microplate reader. MMP-2, MMP-12, HDAC-1, HDAC-6 and metallothionein were obtained from Enzo Life Sciences (Farmingdale, NY). Human carbonic anhydrase, myoglobin and transferrin were obtained from Sigma-Aldrich.

Neutrophils were isolated from the femur and tibia of mice as previously described [12 (link)]. Using a 26-gauge needle and 10 ml syringe filled with RPMI 1640 media supplemented with 1% penicillin/streptomycin and 2 mM EDTA, bone marrow cells were flushed from the bones. The cell suspension was filtered through 30 μm filter to obtain single cell suspension. Single cell suspension was incubated with anti-Ly6G magnetic ultrapure microbeads (Miltenyi Biotec, #130–120–337), and neutrophils were sorted through magnetic columns in the AutoMACS Pro Separator. Purity of Ly6g⁺ neutrophils using this approach has previously been shown to be >95% by both flow cytometry and immunofluorescence [9 (link)]. Neutrophils (1 × 10⁶) were stimulated with IL-1β (200 ng/ml, RnD systems, #401-ML), IL-4 (20 ng/ml, RnD systems, #404-ML), or MMP-12 (500 ng/ml, Enzo Life Sciences #BML-SE138–0010) and incubated for 15 min at 37 °C in 1 ml of RPMI 1640 media. The negative control was unstimulated cells. Samples sizes were n = 3 M and 3 F biological replicates for each stimulus group, performed as a four-group paired stimulation. Following stimulation, cells were centrifuged at 800 xg for 8 min and cell pellets were used for the transcription factor signaling array, while the secretome was used for the cytokine array.