2.1. Cell lines
In this study, we used two cell lines: 1) human H1299 lung cancer cell line from the American Type Culture Collection (ATCC). Culture medium used consisted of RPMI (high glucose, with no L-Glutamine and no Sodium Pyruvate), supplemented with Fetal Bovine Serum (10%), 2 mM L-Glutamine and gentamycin (50 µg/ml). 2) Murine BV-2 microglial cell line (generously provided by Zvi Vogel’s laboratory, Weizmann Institute of Science, Rehovot, Israel): The BV-2 cells were cultured in Dulbecco’s modified Eagle’s medium high glucose containing 4.5 g/l glucose, 4 mM L-glutamine and supplemented with 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/mL) 16. These two cell types were cultured at 37 °C in 5% CO2 and 90% relative humidity.
2.2. TSPO ligands pretreatment
The in vitro experiments included the following groups: vehicle control group pretreated with 1% ethanol-containing 0.5% Fetal Calf Serum (FCS, biological industries, Beit Ha'Emek, Israel); a group pretreated with 1% ethanol-containing serum starvation medium.
The two cell lines were seeded in 96-well plates (5x103 cells per well) or 6-well plates (2.5x105 cells per well) (depending upon the type of experiment) and grown in complete medium for 48 hours at 37ºC and 5% CO2 until the desired confluency 80% was reached. Then, pretreatment with the TSPO ligand PK 11195 (25 µM) in serum-starvation medium was applied for another 24 hours.
2.3. Cobalt Chloride (CoCl2) exposure
CoCl2 (Sigma-Aldrich, Rehovot, Israel) was prepared at the concentrations required for each specific experiment and applied to the CoCl2-treated groups for 24 hours 7,9.
2.4. Cell counting
Cells were grown until 80% confluency was reached, the medium was discarded, cells were washed with phosphate buffer saline (PBS) and collected following trypsinization. For cell counting, 100 µl of the cells was placed in an Eppendorf tube then mixed with 100 µl of trypan blue (ratio 1:1). Under the light microscope, the cells were counted by hemocytometer (Neubauer slide, Sigma Aldrich, Rehovot).
2.5. XTT assay
The two cell lines (H1299 and BV-2) were seeded in 96-well plates (5,000 cells/well) and grown for 48 hours in complete medium. Then pretreated with the required TSPO ligand for another 24 hours, followed by exposure of the cells to the desired concentrations of CoCl2 for 30 minutes, 4 hours, or 24 hours. Assessment of cellular viability was performed using XTT cell viability kit (Biological Industries, Bait Ha'Emek, Isreal), following the manufacturer’s protocol:150 µl medium from each well was removed followed by adding 50 µl from the XTT mixture to the remaining 50 µl medium within the plate, then the plates were incubated in dark for one hour and a half. Reduction of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) depends on mitochondrial dehydrogenases and reductases, which results in orange formazan dye production, a process that occurs only in viable cells. The amount of the orange dye indicates the cellular viability and the optic density (O.D.) was measured using Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland) with absorbance with endpoint photometric at 492 nm wavelength and reference wavelength of 620 nm.
2.6. JC-1 assay
The mitochondrial membrane potential (Δψm) depolarization was assessed using the JC-1 assay, which based on cationic, lipophilic tetra-ethyl-benzimidazolyl-carbocyanine iodide JC-1 dye. After seeding the H1299 cells in 6-well plates (250,000 cells/well) for 48 hours and pretreated with 25 µM PK 11195 for another 24 hours, cells were exposed to 0.5 mM CoCl2 for 24 hours, then cells were trypsinized (600 µl trypsin), collected and centrifuged (660 g for 5 minutes at room temperature) followed by removing the supernatant and resuspended in 600 µl of PBS and cells were centrifuged again. Dilution of JC-1 with PBS (1:500), 600 µl were applied and incubated for 30 minutes in dark. Cells were centrifuged again and 400 µl of PBS were added followed by cells filtration and transfer to FACS tubes. In case of intact cells with high Δψm, JC-1 enters the mitochondria and forms J- aggregates emitting red fluorescence at 590 nm. In contrast, cells exposed to CoCl2, with low Δψm, the JC-1 dye remains in cytosol compartment in a monomer form emitting green fluorescence at 527 nm indicating Δψm depolarization (Zeno et al., 2009). The median fluorescence intensity (MFI) indicates Δψm depolarization that was calculated by red/green ratio using FACS, and the results were analyzed using FlowJo (FlowJo LLC, Ashland, Oregon)
2.7. Nonyl Acridine Orange (NAO) assay- cardiolipin peroxidation indicator
H1299 Cells were seeded in 6-well plates (250,000 cells/well), after 48 hours, 25 µM of PK 11195 was applied for 24 hours, then cells were exposed to 0.5 mM CoCl2 for another 24 hours. Afterwards, cells were trypsinized and centrifuged (660 g for 5 minutes at room temperature) followed by neutralizing trypsin with complete medium. The cells were washed with PBS and centrifuged again the supernatant was aspirated and 400 µl of Nonyl Acridine Orange (NAO) stain (diluted with PBS at a ratio of 1:1000) was added. The cells were incubated in dark for 30 minutes, and centrifuged again, then 400 µl of PBS were added and transferred to FACS tubes.
NAO stain was used to assess the cardiolipin peroxidation level. Cardiolipin, a polyunsaturated acidic phospholipid, biosynthesized and localized in the inner mitochondrial membrane. Cardiolipin is known to have high content of unsaturated fatty acid, which makes it more susceptible to ROS-related cardiolipin peroxidation that results in cytochrome c translocation to the cytosolic compartment, where it initiates the mitochondrial apoptotic cascade (Caballero et al., 2013). NAO staining of the cardiolipin content was performed as described previously (Kluza et al., 2002). Elevated MFI of NAO staining indicates lower cardiolipin content due to increased cardiolipin peroxidation. The MFI was measured using CyAN ADP FACS machine (Beckman Coulter, Brea, CA), and the results were analyzed using FlowJo (10th version, FlowJo LLC, Ashland, OR).
2.8. Necrosis/apoptosis assay
Necrosis/apoptosis assay kit (Abcam, Cambridge, UK) was used to detect apoptosis and necrosis levels in H1299 cells according to the manufacturer’s instructions. Seeding of H1299 cells was performed in 6 well-plates (250,000 cells/well). After 48 hours, PK 11195 at a concentration of 25 µM was applied for 24 hours then followed by 24 hours' exposure to 0.5 mM of CoCl2. Cells were trypsinized using 600 µL trypsin [Trypsin EDTA Solution B (0.25%), EDTA (0.05%)] (Biological industries, Beit Ha'Emek, Israel) centrifuged (660 g for 5 minutes at room temperature) then the supernatant was discarded, and the cells were resuspended in 200 µl assay buffer (provided in the kit) and transferred to Eppendorf tubes. Staining was conducted by adding 2 µl of Apopoxin to 100 µl of sample for apoptotic cells detection, 1 µl of 7-AAD to 100 µl of sample for necrotic cells detection with subsequent incubation in dark for 1 hour. Before reading the samples, 300 µl of assay buffer were added to each sample. Aria FACS machine (BD bioscience, San Jose, CA, USA) was used with Ex/Em = 490/525 nm for detection of apoptosis, Ex/Em = 550/650 nm for detection of necrosis. The 10th version of FlowJo (LLC, Ashland, OR, USA) was used for calculation.
2.9. Cellular ROS/superoxide assay
Cellular ROS/superoxide assay kit (Abcam, Cambridge, UK) was used to detect ROS levels in BV-2 cells upon exposure to 0.3, 0.5, and 0.7 mM CoCl2 for 4 hours and 24 hours, according to the manufacturer’s instructions. On the day of experiment, the medium was removed from wells and washed with 1X washing buffer (provided in the kit in 10X concentration). 100 µl/well of ROS detection dye were applied on the treatment groups for 1 hour in the dark at 37ºC. Bottom reading of the plates using Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland) was performed with Ex/Em= 488/520 nm.
2.10. Caspases multiplex activity assay
Assessment of the apoptotic markers caspase 3 and caspase 9 was performed in BV-2 cells using the caspase multiplex activity assay kit (Abcam, Cambridge, UK). BV-2 cells were seeded in 96-well plates (104 cells/90 µl) and pretreated as required. Following the cells exposure to the desired concentrations of CoCl2 (0.5 and 0.7 mM) for 4 hours. The diluted caspases 3 and 9 substrates (diluted in assay buffer at ratio of 1:200) were added to the cells and incubated for one hour. Measurements of fluorescence intensity at Ex/Em of 535/620 nm for caspase 3, and at Ex/Em of 370/450 for caspase 9 were conducted using the Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland).
2.11. TSPO protein expression levels
Measurement of TSPO protein levels in BV-2 cells was performed using FACS upon 4 hours' exposure to 0.3, 0.5, and 0.7 mM CoCl2. Medium with the cells were collected from each sample and centrifuged (660 g for 5 minutes at room temperature), then supernatant was removed. Cells were washed using PBS and incubated with PBS containing 0.2% Tween 20 (PBS-T) for 10 minutes and centrifuged again at 660 g for 5 minutes at room temperature. Then, the cells were incubated overnight with primary anti-TSPO antibody (Abcam, Cambridge, UK) diluted with ratio 1:100 in 3% BSA in PBS-T. On the following morning, the cells were washed with PBS, incubated with goat anti-rabbit AlexaFlour488 secondary antibody for 1 hour, then washed and transferred to FACS tubes. MFI was measured using FACS machine. The results were analyzed using FlowJo 10th version (LLC, Ashland, OR, USA).