Bone marrow cells were collected from tibias and femurs of age- and sex-matched 8- to 12-week-old mice described in the “Mice” section and cultured in either complete Dulbecco’s Modified Eagle’s Medium (DMEM) [supplemented with 10% fetal calf serum, 10 mM glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml), and 50 μM 2-mercaptoethanol] with 20% L929 conditioned medium for 6 days to generate BMDMs or complete RPMI (same supplements as for complete DMEM) containing GM-CSF (15 ng/ml) for 8 days to generate BMDCs.
Adenosine triphosphate (ATP) cell viability assays were conducted using the CellTiter-Glo Luminescent Cell Viability Assay Kit (G7570, Promega, Fitchburg, WI). Luminescence was quantitated using the Victor3 1420 Multilabel Counter (PerkinElmer, Shelton, CT). Cell death was also determined by PI staining (33 (link)) and quantitated under fluorescence microscopy or by using a flow cytometer. Alternatively, lactate dehydrogenase (LDH) release was used to determine the extent of cell death using Cytotoxicity LDH Assay Kit-WST (CK12, Dojindo Molecular Technologies Inc.).
Adenosine triphosphate (ATP) cell viability assays were conducted using the CellTiter-Glo Luminescent Cell Viability Assay Kit (G7570, Promega, Fitchburg, WI). Luminescence was quantitated using the Victor3 1420 Multilabel Counter (PerkinElmer, Shelton, CT). Cell death was also determined by PI staining (33 (link)) and quantitated under fluorescence microscopy or by using a flow cytometer. Alternatively, lactate dehydrogenase (LDH) release was used to determine the extent of cell death using Cytotoxicity LDH Assay Kit-WST (CK12, Dojindo Molecular Technologies Inc.).