Fascalibur

The effect of compounds 7i and 7k on the polymerization of purified brain tubulin was determined employing a fluorescence-based tubulin polymerization assay kit (Cat. #BK011P, Cytoskeleton, Inc., USA) according to the manufacturer’s protocol. Tubulin was re-suspended in ice-cold G-PEM buffer (80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP, 20% (v/v) glycerol) and added to wells on a 96-well plate containing the designated concentration of drugs or vehicle. Samples were mixed well, and tubulin assembly was monitored (emission wavelength: 450 nm; excitation wavelength: 360 nm) at 1 min intervals for 90 min at 37°C using a plate reader (FASCalibur, BD Biosciences, USA). IC₅₀ values were calculated at 80 min using SPSS software in three experiments.

Cells without treatment or after treatment with the indicated drugs overnight were harvested in PBS and incubated in the presence of 10 μM 2’7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Invitrogen) in the dark at 37°C for 30 minutes. The shift in green fluorescence was measured in a FASCalibur flow cytometer (BD Bioscience) using the FL-1 detector and ROS production was determined from histogram data using CellQuest software (BD Bioscience). A total of 20,000 events were collected for each histogram.

HA1800 astrocytes were dissociated into single cells using TrypLE and then washed with cold PBS containing 0.5% BSA and 2 mM EDTA. Subsequently, the astrocytes were incubated with FITC-conjugated mouse anti-human CD44 or FITC-conjugated mouse IgG2a, κ isotype control (BD Bioscience, USA) for 30 min at 4 °C in the dark before being washed 3 times with cold PBS buffer. FASCalibur (BD Biosciences) and Flowjo software were used to acquire and analyze data.

Apoptosis was evaluated using the terminal‐deoxynucleotidyl transferase–mediated nick‐end labelling (TUNEL) assay and flow cytometry (FAS Calibur, BD). According to the manufacturer's instructions, httex1p‐Q74 cells were collected and incubated with Annexin V‐PE and 7‐aminoactinomycin D (7‐AAD) (Southern Biotech, Birmingham, AL) in binding buffer. Annexin V+/7‐AAD– cells were identified as early apoptotic, while Annexin V+/7‐AAD+ cells were identified as late apoptotic. Apoptosis was observed by TUNEL staining using an in situ cell death detection kit (Roche, Mannheim, Germany). The average number of TUNEL‐positive cells was calculated in five randomly selected regions.

Cell death was detected using the Multi-Parameter Apoptosis Assay Kit (Cayman Chemical). Briefly, cells were counted and seeded in 96-well or 6-well plates. Cells without treatment or after treatment with the indicated drugs overnight were then washed with Cell-Based Assay Binding Buffer, and then incubated with Annexin V-FITC/7-Amino-Actinomycin D (7-AAD) double staining solution at room temperature in the dark for 10 minutes before resuspending in binding buffer for analysis. Fluorescence intensities were analyzed using SpectraMax M5 multi-detection microplate reader (Molecular Devices, Sunnyvale, CA) or on a FASCalibur flow cytometer (BD Bioscience, San Jose, CA) under FL1 (Annexin V-FITC) and FL3 (7-AAD) detectors. Annexin V-positive cells are defined as apoptotic, and Annexin V-7-AAD-double positive cells are defined as necrotic for flow cytometric analysis.

Cells were detached with trypsin/EDTA solution to obtain single-cell suspension. Further, cells were washed with 1% FBS in PBS and subsequently stained with antibodies. Primary antibodies were FITC conjugated anti-human CD44 (BD Bioscience Pharmingen, USA), APC-conjugated anti-human CD90 (Immuno Tools, Germany), PE-conjugated anti-human CD105 (Immuno Tools) and PerCP-conjugated anti-CD45 (Immuno Tools). Stained cells were analysed using a FASCalibur using the CellQuest software (BD Bioscience, USA).

CD4 and CD8 T Lymphocyte counts were performed using a FacsCalibur (FASCalibur, BD, Biosciences, San Jose, CA, USA). Whole blood was stained using a lyse/wash immunophenotyping procedure. One hundred microliter of peripheral whole blood was added into a polystyrene tube containing 20 μL of a monoclonal antibody cocktail of anti-CD3 Fluorescein isothiocyanate (FITC), anti-CD8 Phycoerythrin (PE), anti-CD45 Peridinin chlorophyll protein coupled to the cyanine dye Cy™ 5.5 (PercCp-Cy5.5) and anti-CD4 allophycocyanin (APC). Cells and antibodies were then incubated in dark for 15 min at room temperature. Thereafter, 2 mL of lysis buffer (BD Biosciences, San Jose, CA, USA) was added to the cells and kept for 10 min in dark at room temperature to allow red cell lyses.
Cells were then washed twice with 2 mL of BD Wash Buffer (Becton Dickinson) and re-suspended in 200 μL of BD Wash Buffer for acquisition with a four-color flow cytometer (FASCalibur, BD Biosciences, San Jose, CA, USA). Data were later analyzed with FlowJo software (Tree Star inc. Ashland, Oregon, USA).