Mice were sacrificed after exposure to saline or Be(OH)2, and BAL was performed with 1.5ml of sterile PBS. Cells were separated from the BALF using centrifugation, and viable cells were enumerated using trypan blue. Beryllium crystals have a crystalline morphology that can be clearly distinguished from viable cells. For IL-1 analysis BALF were concentrated 5-fold using Amicon 3kDa filters. IL-1α and IL-1β were quantified using Ready Set Go ELISA (eBioscience).
BAL cells were incubated with FV-eFluor 506 for 30 min on ice (eBioscience), washed, incubated with Fc Block and mAbs directed against Ly6G, Siglec F, F4/80 and Ly6C. Live and dead cells were gated based on unstained controls (Fig S1A ). Live cells were analyzed for the presence of neutrophils and macrophages and for apoptotic cells using the FITC Annexin V kit (eBioscience). Data were acquired on a LSRII SORP flow cytometer using FACS Diva Software (BD Biosciences) and analyzed using FlowJo software (Treestar, Inc.).
BAL cells were incubated with FV-eFluor 506 for 30 min on ice (eBioscience), washed, incubated with Fc Block and mAbs directed against Ly6G, Siglec F, F4/80 and Ly6C. Live and dead cells were gated based on unstained controls (