Mmp200

Peripheral blood samples of ApoE KO mice were collected from the postcaval vein using a 24-gauge catheter (382412, BD) at 28 days. The blood plasma was separated using Ficoll-Paque plus (#17–1440-03, GE). Circulating IL-6 (M6000B), TGFβ (MB100B), IL-10 (M1000B), tumor necrosis factor (TNF) α (MTA00B), intercellular adhesion molecule (ICAM; MIC100), MMP-9 (MMPT90), vascular cell adhesion molecule (VCAM; MVC00), and MMP-2 (MMP200, all from R&D systems) levels were evaluated using mouse ELISA kits. The ELISA reaction product was quantified by measuring absorbance at 450 nm and 540 nm using a microplate reader (iMark), and data were analyzed using Microplate Manager software (Ver. 6.1, both from Bio-rad).

MMP-14, TIMP-2 and total MMP-2 tissue levels were quantified using sandwich enzyme-linked immunosorbent assay (ELISA) kits. A standard curve was run in each assay; all samples and standards were measured in duplicate and findings averaged. The TIMP-2 assay procedure was done according to the manufacturer’s instructions (DTM200 and MMP200, R&D Systems). The MMP-14 assay procedure was carried out according to the manufacturer’s instructions with the incubation time of the samples with the antibody cocktail increasing to two hours (ab197747, Abcam).

Brain tissues were homogenized in RIPA buffer (MilliporeSigma) containing complete protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). After centrifugation (100,000g for 1 hour at 4°C), the supernatant was used for biochemical analysis as described (53 (link)). ApoE (53 (link), 55 (link)), collagen IV (LS-F20750, LSBio), MMP-2 (MMP200, R&D Systems), and MMP-9 (NBP2-60095, Novus Biologicals) in brain lysate were measured by ELISA. Brain lysate measurements were normalized to total protein concentrations determined by BCA assay (Thermo Fisher Scientific). Total cholesterol and triglycerides in plasma were measured using a Cholesterol Assay Kit (A12216, Thermo Fisher Scientific) and Triglyceride Assay Kit (ab65336, Abcam), respectively. Some brain samples were subjected to Western blotting using anti–Iba-1 antibody (17198, Cell Signaling Technology) and anti–β-actin antibody (3700, Cell Signaling Technology), followed by quantification through LI-COR Odyssey.

We analyzed the expression of type I IFN, CXCL16, and MMP-2 using the following ELISA kits: type I IFN (MIF00, R&D systems), CXCL16 (ab100677, Abcam, Cambridge, UK), and MMP-2 (MMP200, R&D systems). We analyzed the expression of 144 cytokines in the supernatants of cocultured B6 mouse spleen cells and iPS-ML or iPS-ML-41BBL. The B6 mouse spleen tissues were harvested and dissociated into single-cell suspensions using the gentleMACS Dissociator (Miltenyi Biotec). Red blood cells (RBCs) were lysed using RBC lysis buffer (Roche). The spleen cells (1 × 10⁶ cells/well) and iPS-ML or iPS-ML-41BBL (1 × 10⁵ cells/well) were cocultured for 5 days in 6-well culture plates in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL recombinant mouse IL-2 (R&D Systems), and 50 μM 2-mercaptoethanol. The supernatants were then sampled and assayed using the mouse cytokine array C-series 2000 kit (Ray Biotech, Norcross, GA USA).