Rat macrophage colony stimulating factor (M-CSF) was purchased from GenScript (Piscataway, NJ). ELISA kits were obtained from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS) from Escherichia coli OIII:B4 was purchased from Invivogen (San Diego, CA; #LPS-EB). All assays and techniques were performed according to the provided guidelines.
Spontaneously Hypertensive Stroke Prone (SHR-SP) and Sprague Dawley (SD) rats
Male and female SHR-SP rats were used to produce the BMDM. Timed pregnant SD rats were used in the production of microglia and were purchased from Taconic (Hudson, NY).
Bone marrow derived macrophages
BMDM were derived from the femurs/tibias of SHR-SP rats as has been previously described. DMEM supplemented with 10% FBS and 10 ng/ml rat M-CSF was used to culture the cells. Briefly, a 100 mm tissue culture dish was seeded with 2×106 cells in a total of 11 ml culture medium. After 3 days, 5.5 ml medium with 30 ng/ml rat M-CSF was added to each dish. After 6 days in total, PBS was used to wash the cells twice prior to scraping in cold HBSS. After centrifugation and resuspension, the cell density was adjusted to 2.5×105 cells per ml and 2 ml cells were deposited in each well of six-well-plates. Iba-1 staining confirmed that >99% of the cells were macrophages. On the 7th day, the cells were exposed to OGD or OGD + LPS (10 ng/ml). Supernatants from OGD/LPS-treated cells were collected and used for ELISA.
The isolation of cells was performed as previously described with minor modifications. Briefly, 2–4 day old pups (determined via the use of timed pregnant SD rats) were used to make primary microglia. After the brains were dissected, the meninges were removed and the cortices placed in cold HBSS. After treatment with papain and DNase (Worthington Biochemical, Lakewood, NJ) for 15 min at 37°C, the cells were triturated 10x followed by centrifugation at 300 g for 5 min. The cells were then resuspended in DMEM plus 10% FBS and 10% horse serum (Life Technologies, Frederick, MD) and seeded at 2.5×105 cells per ml in poly-L-lysine (Sigma, St. Louis, MO) coated flasks; the medium was changed the following day. Microglia within the supernatant were collected 4–6 days later, plated on poly-L-lysine coated plates, and used experimentally on the following day.
Oxygen and glucose deprivation
Prior to OGD cells were washed twice with PBS and 1 ml culture medium without glucose (Life Technologies, Grand Island, NY), 1 ml of the glucose-free media was subsequently added to each well. The plates were then placed within modular incubator chambers (Billups-Rothenberg, San Diego, CA) with an anaerobic indicator strip capable of detecting a 0.2% oxygen threshold (Becton Dickinson). The chamber was flushed with a gas mixture containing 95% N2 and 5% CO2 at 6 L/min for 20 min at room temperature, sealed and placed at 37°C.
Commercial IP lysis buffer (Thermo Fisher Scientific, Rockford, IL) was added directly to each well of the six-well plates to prepare cell lysates. The lysate was incubated for 15 min on ice and subsequently underwent 5 s of sonication. The lysate was then centrifuged at 10,000 g for 15 min at 4°C. The supernatant was collected and a BCA assay (Thermo Fisher Scientific, Rockford, IL) was used to determine the protein concentration. The samples were then heated for 5 min at 95°C, and 15 μg of the total cell lysate was used for SDS-PAGE. The following primary antibodies were used for the western blot analyses: anti-caspase-1 (Abcam, Cambridge, MA; #ab108362), anti-NLRP3 (AdipoGen, San Diego, CA; #AG-168 20B-0014), anti-ASC (AdipoGen, San Diego, CA; #AG-25B-0006), anti-AIM2 (Santa Cruz Biotechnology, Santa Cruz, CA; #SC137967). Signals were detected using a chemiluminescent substrate (Immobilon Western; Millipore, Billerica, MA) followed by digital imaging (Alpha Innotech, San Leandro, CA) or C-Digit (LI-COR, Lincoln, NE).
All data are expressed as mean ± SD of three independent experiments. Western blot and ELISA data were compared using the Student’s t-test. Differences were considered significant when p <0.05.