Magnetic holder

Mice were sacrificed 1, 3, or 7 days post-ICH modeling after perfusion with isolation medium (HBSS containing 0.05% glucose, 15 mM HEPES, and 0.2% DNase I). Brains were dissected from the mice rapidly, brain slides were cut into 1 mm thickness, and the perihematoma tissues were isolated. Collected tissues were chopped and homogenized in 3 ml of cold isolation medium using a Dounce homogenizer to generate single cells suspension. The suspension was rinsed by another 2 ml medium after filtered through a 100-μm cell strainer. Following 340 × g centrifuge for 5 min, the precipitate was resuspended in 30% Percoll and centrifuged at 900 × g for 20 min with lowest lifting speed to remove myelin [25] (link). To further purify microglia, precipitated cells were resuspended in MACS buffer and wash twice, then 10 µl of CD11b MicroBeads (Miltenyi Biotec) was added per sample and incubated for 15 min in room temperature. After resuspension, cells were transferred into an LS separation column which placed in a magnetic holder (Miltenyi Biotec) and labeled cells remained in the magnetic field. To gain the CD11b positive cells, carefully removed column from the magnetic field and washed the column with MACS buffer into collection tubes.

We routinely applied 2 × 10⁵ PMNs per milliliter to cells. For antibody-mediated clustering of ICAM-1, sheep anti-mouse IgG-coupled dynabeads (M280; Invitrogen) were coated with mouse anti-human ICAM-1 antibody (BBIG-I1; R&D Systems) or immunoglobulin IgG1 (IgG1) control (MAB002; R&D Systems) overnight at 4°C according to the manufacturer’s protocol. To induce clustering, 1.5 × 10⁶ antibody-coated beads/mL was added to a tumor necrosis factor α (TNFα)-stimulated HUVEC monolayer cultured in a 6-well dish and incubated for 15 minutes. For some experiments, antibody-coated beads (5 × 10⁵/mL) were injected into the ibidi perfusion system containing HUVECs to induce ICAM-1 clustering on HUVECs under physiologic flow conditions. Alternatively, ICAM-1 was ligated with 15 μg/mL mouse monoclonal antibodies (R&D Systems, catalog no. BBIG-I1) for 30 minutes, followed by washing and ICAM-1 cross-linking with 50 μg/mL mouse secondary antibody (R&D Systems, catalog no. AF007) for 20 minutes at 37°C. Live cell imaging of membrane tension and intracellular Ca²⁺ during ICAM-1 clustering were performed using a Leica SP8 or an Olympus IX81 microscope (see above). For immunoblot analysis, beads were isolated using a magnetic holder (Miltenyi Biotec), and cells were lysed with RIPA buffer as described above.