Fitc conjugated anti cd40

To measure the effect of TGF-β1 on microglial surface marker expression after 3-AP infusion, flow cytometry was conducted. On day four after TGF-β1 injection, rats were anesthetized with sodium pentobarbital (55 mg/kg) and perfused with cold PBS via the left ventricle of the heart to eliminate cells in the vasculature. Brain stem and cerebellum tissues were isolated, respectively, and digested with collagenase D (2.5 mg/ml; Roche, Penzberg, Germany) and DNaseI (1 mg/ml; Sigma-Aldrich, St. Louis, MO, United States) at 37 °C for 20 min. Then, the single cells were collected by passing the tissues through a 70 μm cell strainer, followed by a percoll gradient (25 and 75%) centrifugation. The mononuclear cells at the 25/75 interphase were obtained for subsequent analysis. The cells were labeled with APC-conjugated anti-CD11b, FITC-conjugated anti-CD40 or PE-conjugated anti-CD86 (all from eBioscience, San Diego, CA, United States) or the appropriate isotype control antibody. The frequency of the respective immunoreactive cells was expressed as a percentage in total mononuclear cells using a FACS Calibur flow cytometer (BD Biosciences, United States) equipped with CellQuest software.

Bone marrow-derived dendritic cells were stained in FACS buffer (PBS containing 0.1% bovine serum albumin and 5 mM EDTA) using the following monoclonal antibodies in the indicated concentration for cell surface markers (all obtained from eBioscience): PE-conjugated anti-CD86 (2 μg/ml), FITC conjugated anti-CD-40 (5 μg/ml), PE-conjugated anti-CD11c (1 μg/ml), and APC-conjugated MHC-II (0.28 μg/ml). Cells were first incubated with Fc receptor block, 5 μg/ml (eBioscience) for 10 min to block any non-specific binding and subsequent staining steps were performed for 20 min at 4°C, followed by washing with FACS buffer. An isotype-matched control staining was done for each antibody to determine a specific background staining. Cells were acquired using a Cyan-ADP cytometer (Beckman Coulter, Woerden, The Netherlands) and analyzed with FlowJo software (FlowJo LLC, Ashland, OR, USA).

BV2 cells were stained with FITC-conjugated anti-CD40 (eBioscience, San Diego, CA, USA) and Pe-Cy5-conjugated anti-CD86 (eBioscience, San Diego, CA, USA). Appropriate isotype control antibodies were used where necessary to set gates for cell marker positivity. Typically, proportion of isotype control antibody-stained cells was <1%. Detection of apoptosis was performed via annexin V-FITC staining (Biotium, Hayward, CA, USA). Cells positive for annexin V-FITC were considered apoptotic. For detection of phagocytosis, BV2 cells were plated in 24-well plates at 1 × 10⁵/well and pretreated with GYY4137 for 24 h. Latex beads (1 μm, yellow-green) were preopsonized in PBS supplemented with 50% FCS. Medium was discarded and the preopsonized beads were added (10 beads per cell), and cells were incubated at 37 °C for an additional hour. BV2 cells were analyzed with a CyFlow Space flow cytometer (Partec, Munster, Germany). Results of cytofluorimetry are presented as the proportion of cells bound by an appropriate antibody or as mean fluorescent intensity (mfi) of cell population. Cells were gated on live cells (R1) according to FSC vs. SSC and on cell singlets (R2) according to FSC vs. FSC-width, and mfi (arithmetic mean) was determined for all the cells within G1 (R1 and R2) by FloMax software for cytometry (Partec, Munster, Germany).