Annexin 5 fitc

Flow cytometry was performed with a LSRII machine (Becton Dickinson) to analyze T-cell developmental and cell death markers from healthy Lck-Dlx5 mice. CD4-APC/Cy7, CD8-PE, CD44-APC/Cy7, CD25-PE, Notch1-Alexa647 and Notch3-APC antibodies were obtained from BioLegend. Annexin V-FITC and ethidium homodimer III were from Biotium. Data were analyzed with FlowJo software.

The N2a cells were grown on coverslips, coated with poly D-lysine (0.1 mg/mL) in minimum essential medium (MEM, GIBCO) containing 10% heat-inactivated fetal bovine serum (FBS) and pen/strep (1 μg/mL). Prior to the experiment, the media were replaced with fresh MEM, which lacked growth factors, and treated with Aβ42 (10 μM), along with different doses of Cur and/or SLCP (in μM: 1, 0.1, and 0.01), dissolved in methanol, and diluted with fresh MEM for 24 h. Then coverslips were washed in PBS twice, stained with Annexin-V-FITC (apoptosis detection kit; Biotium, Hayward, CA), as per manufacturer's instruction. The images were taken using a fluorescence microscope (Leica, Germany) with blue/green/red filters. The total number of cells and the numbers of apoptotic cells were counted per microscopic field and expressed as a percentage of dead neurons. At least 30 microscopic fields from three independent experimental setups were used for counting.

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).