The following antisera were used in these studies: anti-IDE antibodies 9B12 (Abcam) and 402038, anti-LDH antibody Ab 52488 (Abcam), monoclonal anti-GAPDH (6C5) (Santa Cruz), rabbit antibody against the human pitrilysin sequence 753AEMTDIKPILRKLPRIKK (Bethyl Lab)38, monoclonal anti-Bip/GRP 78 (BD Transduction Laboratories), monoclonal anti-β-actin (C4) (Santa Cruz), monoclonal anti-Alix (1A12) (Santa Cruz), and monoclonal anti-flotillin-1 (BD Transduction Laboratories). Fetal bovine serum was purchased from Atlanta Biologicals. Two different anti-IDE antibodies, 9B12 and 4020, were used because the 9B12 monoclonal antibody, raised against human erythrocyte IDE, did not react very well with rodent IDE (BV2 and Neuro2a cells) in Western blots but did react well with human IDE in Western blots (HEK293 cells; Supplementary Fig. S7). The 4020 polyclonal antibody, rasied against rat IDE, reacted well in Western blots with rodent IDE ((BV-2, Neuro2a cells) but not well with human IDE (HEK293 cells). IDE release was calculated from data obtained with one or the other of these antibodies.
Cell Culture and Protein Measurements
HEK293 cells (ATCC) were grown overnight in 10 cm dishes at 37 °C with 5% CO2 to ~75% confluence in 10 ml DMEM media (Gibco by Life technologies) containing 10% FBS. The culture media was carefully removed and 10 ml of fresh media was added. At the desired time points the plate was briefly removed from the incubator and a 0.25 ml aliquot of the media taken for analysis of secreted IDE. At the end of the incubation period the cells were washed with 10 ml of cold PBS, and 4 ml of RIPA buffer was added. After a 5 min incubation, the RIPA buffer was collected and sonicated for 20 sec with a 40% pulse using a Branson Digital Sonifier. The sonicated solution was centrifuged for 10 min at 20,000 g and used for measuring total cellular IDE and LDH. In preliminary experiments, separate cultures were used for each time point. This yielded essentially the same results, but with more scatter in the data.
Secreted IDE and LDH were measured by SDS-PAGE, running 50 µL of each sample on a 10% polyacrylamide gel. IDE was measuring using anti-IDE antibody 9B12 for HEK293 cells or anti-IDE antibody 4020 for BV-2 and Neuro2a cells, while LDH was measured using anti-LDH antibody 52488. Total cellular IDE and LDH were measured on the same gel using a 2.5 or 5.0 µL aliquot of the RIPA buffer homogenate. Bands were visualized using SuperSignal West Pico (Thermo Scientific) or SuperSignal West Femto (Thermo Scientific), scanned on a ChemiDoc MP System (BioRad), and analyzed with Image Lab software (Bio-Rad Laboratories, Inc.). Linearity of Western blot response was verified using recombinant purified IDE (Supplementary Fig. S8), and scans were taken at multiple exposure times to insure linearity. The sensitivity of these assays was very similar, with the LDH assay being slightly more sensitive than the IDE assay.
The percentage of IDE or LDH in the conditioned media was calculated as given in equation (1).
where %CM is the percentage of enzyme in the conditioned media, TCM is the total scanned Western blot density from conditioned media, and TCL is the total scanned Western blot density from the cell lysate.
BV-2 cells, developed by Blasi et al.39 (obtained from Dr. Elisabetta Blasi, University of Perugia), were grown overnight at 37 °C with 5% CO2 to ~50% confluency in 60 mm dishes containing 4 ml of DMEM media (GibCo by Life technologies) and 10% FBS. The next day the media was changed to DMEM with N2 supplemented media (GibCo by Life technologies) with and without 10% FBS or RPMI 1640 media (GibCo by Life technologies). Following overnight growth the media was changed and cells were incubated for 0, 4.5, 9, and 18 h. At each time point an aliquot of the conditioned media was carefully removed and centrifuged at 2,500 × g for 5 min. to remove cell debris. This conditioned media was used to assess the presence of IDE and LDH while a cell lysate was prepared as described above.
Neuro2a cells (ATCC) were grown in 50% DMEM/50% Opti-MEM media containing 5% FBS, and samples for secreted IDE and LDH were taken and analyzed as described for the other cell types.
BV-2 cells grown in RPMI were treated with 5 µM lovastatin (Axxora, Enzo Life Science) for 24 h. The conditioned media was collected and assayed for the presence of IDE, LDH, GAPDH, and pitrilysin as noted above.
Cell viability assays
For trypan blue analysis, cells were seeded in quadruplicate at ~50% confluency into 6-well plates, cultured in a 5% CO2 incubator at 37 °C for 24 h. In some cases lovastatin was added to a final concentration of 5 µM and cells were incubated for an additional 24 h. After the incubation period cells were treated with 1/10 of the original volume of 0.5% trypsin/EDTA for ~5 min. The detached cells were then diluted ten fold with a 0.4% trypan blue solution prepared in PBS. Following an ~5 min incubation period cells were counted using a TC10™ Automated cell counter, (Bio-Rad, Inc.). Alternatively the trypsinized cells (~100 cells) were manually counted and scored for the number of cells that had taken up trypan blue. Both methods produced similar results.
Cell metabolism was measured with the MTS assay using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions. This assay measures the activity of NADPH dependent dehydrogenases. Cells at ~50% confluency were seeded in quadruplicate in a 24-well plate and cultured as described above. The absorbance at 490 nm was measured with a microplate reader (SpectraMax M5, Molecular Devices).
Isolation and Analysis of Exosomes
Exosomes were isolated as described by Bulloj et al.6. BV-2 cells were grown overnight at 37 °C with 5% CO2 to ~50% confluency in 7 ml of DMEM media containing 10% FBS. The next day the media was replaced with fresh media or with fresh media containing 5 µM lovastatin and incubated for 24 h. The conditioned media was then collected, centrifuged at 800 × g for 10 min at 4 °C to pellet the cells. The cell free conditioned media from 6 plates was combined to yield ~40 ml which was centrifuged at 12,000 × g for 30 min. at 4 °C to produce a pellet (P2). The supernatant from the P2 fraction was centrifuged at 100,000 × g for 2 h at 4 °C to pellet exosomes (P3). The pellets (P2, P3) were resuspended in 100 µl of PBS. These fractions as well as the supernatant (S3) were analyzed by Western blots using the appropriate antisera. Scans were taken for multiple exposure times to establish linearity.