Mirvana lysis buffer

Exosomes were isolated by ultracentrifugation using cell culturing medium from EML4-ALK positive (H2228) and wild type control (A549) NSCLC cell lines. The medium and plasma was first cleared of debris by centrifugation; twice at 500xG for 10 minutes, followed by twice at 2,000xG for 15 minutes, and lastly twice at 10,000xG for 30 minutes. The exosomes in the cleared sample were pelleted at 100,000xG for 60 minutes. The exosomes were washed once in PBS before either being labeling for uptake experiments or DNAse-I treated (4 units; Promega) for 15min before lysis in miRVANA lysis buffer (Ambion) for RNA extraction (according to manufacturer's recommendations) for mutation detection experiments. Exosome uptake was analyzed using PKH67-labeled (Sigma-Aldrich) H2228 microvesicles and the LSM-710 confocal microscope system with the ZEN 2010 software (Carl Zeiss) and a 63x oil immersion objective (Carl Zeiss) as previously described [14 (link)]. Platelets were stained with rhodamine phalloidin (Invitrogen) to indicate platelet structure and analyzed for exosomal uptake by the presence of green fluorescent dye PKH67 (Sigma-Aldrich). EML4-ALK rearranged RNA within platelets was shown by PCR on reverse transcribed RNA with the same setup as previously described.

To generate BMDM, the bone marrow cells from femurs and tibias from mice were harvested and cultured as previously described [5 (link),28 (link)]. Briefly, isolated cells were incubated in Dulbecco’s Modified Eagle Media (DMEM, Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Grand Island, NY)), 1% penicillin/streptomycin, 1% glutamine, and 20% L929 cell supernatant (containing macrophage colony stimulating factor). On day 7 in culture the cells were washed, counted and replated in DMEM media (without L929 supernatant) at a density of 6-8x10⁶ cells/well (6-well plate, Falcon polystyrene). Cells were classically activated (M1 condition) with LPS (100 ng/ml, Sigma-Aldrich L2880) + IFN-γ (20ng/mL, eBioscience, San Diego, CA), alternatively activated (M2 condition) with IL-4 (20ng/mL, eBioscience) or received media alone (M0 condition). The LPS+ IFN-γ condition is used to simulate infectious and/or autoimmune conditions in which Th1 cells produce IFN-γ while pathogens or tissue damage provide PAMPs or DAMPs, respectively. Cells were harvested at the indicated time points, generally 24 hours post-stimulation, by washing in phosphate buffered saline (PBS) before cell lysis in miRVana Lysis buffer (Life Technologies) for total RNA isolation.