Fetal calf serum (fcs)

Immortalized C57BL/6J mouse embryonic fibroblasts (MEFs; kindly provided by Dr. D. Stetson) and HEK293T were cultured in DMEM supplemented with 10% fetal calf serum (VWR), L-glutamine, penicillin/streptomycin, sodium pyruvate, HEPES and β-mercaptoethanol. For lentiviral transduction, the ORF for mouse Sucnr1 was obtained directly from prepared cDNA from sorted small intestinal tuft cells (described above). After sequencing verification, mSucnr1 was cloned into the pRRL-MND-MCS-2A-Puro lentiviral vector downstream of an MND promoter as previously described (Gray et al., 2016 (link)). VSV-G pseudotyped, self-inactivating lentivirus was prepared by transfecting HEK293T cells with 1.5 μg pVSV-G, 3 μg psPAX-2, and 6 μg pRRL lentiviral vector, and viral supernatant collected and filtered before use. MEFs were incubated with viral supernatant overnight, and media was replaced with fresh culture media. Puromycin (5ug mL⁻¹) selection was added 72 hours post transduction and continued for 7 days.

Bone marrow cells were isolated from femurs and tibias of matched 12-week old female WT and Aldh1a1^−/− mice as described above. Bone marrow cells (4.5×10⁵ cells/well) were then plated in a 96-well dish in differentiation medium consisting of αMEM supplemented with 10% fetal calf serum (VWR), 1% Penicillin-Streptomycin 1000U (Invitrogen), 30 ng/mL macrophage colony-stimulating factor (m-CSF) and 100 ng/mL soluble receptor activator of nuclear factor-κB ligand (sRANKL) (PeproTech, Inc.). When indicated, cells were treated with either 1μM of Rosiglitazone (Cayman Chemicals), Rald, (Sigma Aldrich), or the combination of Rosiglitazone and Rald. The culture medium and treatments were changed at day 3 and day 6. At day 7, the culture was terminated; cells were then fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences) and stained for tartrate-resistant acid phosphatase activity (TRAP5b) using the Sigma Leucocyte acid phosphatase (TRAP) kit. Osteoclasts were identified by TRAP5b positive multinucleated cells (more than four nuclei) using light microscopy.

Human umbilical vein endothelial cell (HUVEC) and human arterial endothelial cell (HAEC) cultures were obtained from ATCC (Leesburg, VA) and grown in full EBM-2 medium (Lonza, Portsmouth, NH). Cells were cultured at 37°C in the atmosphere of 5% CO₂. Cells at passages 5 to 15, at 90–100% confluency were used for TXA studies. HL60/S4 cell culture was a gift of Ada and Don Olins (University of New England, Portland, ME), and grown in RPMI medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (VWR, Radnor, PA) at 37°C in the atmosphere of 5% CO₂. To differentiate HL60 cells to granulocytes, they were treated for 72 h with 1 µM all-trans-retinoic acid (Sigma-Aldrich, St Louis, MO). The differentiation of HL60 cells was confirmed by the analysis of nuclear morphology after DAPI staining (development of nuclear segmentation), and by the positive staining with nitroblue tetrazolium. NIH3T3 cells were obtained from ATCC and cultured in DMEM with 10% fetal calf serum. Mito-QC mice were a gift from Anyonya Guntur (MMCRI, Scarborough, ME). Lung EC were isolated from 7-days-old pups of mitoQC mice using two cycles of magnetic separation based on CD31 and ICAM cell surface positivity, as described [Sobczak et al., 2010 ] . Lung EC were cultured in full EBM-2 medium. Immunofluorescence staining demonstrated that at least 90% of the isolated EC were CD31-positive.