Fcs express 4

The variation in ΔΨm was investigated using the BD^™ MitoScreen Kit (BD) according to the manufacturer's instruction. Briefly, 1 × 10⁶ cells were harvested from the suspension cultures, washed twice with PBS and incubated with JC-1 solution for 15 min at 37°C. JC-1 monomers or aggregates were analyzed by flow cytometry (BD AccuriTM C6 cytometer and FCS Express 4 Software).

The number of microglia/macrophage, their proliferation properties and inflammatory cytokine secretion were analyzed by flow cytometry. Single cell suspensions prepared from brain tissues were stained with fluorescently labeled antibodies: APC-CD45, PerCP-Cy5.5-CD45, PE-Ly6G, BV421-Ly6G, PE-Cy7-CD11b, PerCP-Cy5.5-BrdU, PE-IL-1β, V450-IL-6, or APC-TNF-α at designed combination. All antibodies and the isotype controls were purchased from BD Biosciences, San Jose, CA, USA. The staining was performed according to the manufacturer’s instructions and the details are available in Additional file 1: Supplementary Methods. After staining, samples were analyzed using a FACSAria Ι flow cytometer (BD Biosciences). To avoid the interference of GFP expressed on CX3CR1^-/- microglia/macrophage, fluors such as FITC and Alexafluor 488 that excite in the same spectrum at the same filter sets as GFP were excluded and the proper compensation was performed in flow cytometry. Subsequent data analyses were completed using FACSDiva software or FCS Express 4 software (BD Biosciences).

Cells were washed with complete RPMI medium and diluted to a concentration of 1 × 10⁶ cells/100 μL. Cells in the 2 aliquots (100 μL/aliquot) were pelleted by centrifugation at 1800g for 8 minutes and incubated with 100 μL of PBS solution and optimal concentrations of fluorescein isothiocyanate‐conjugated CD4⁺ (Mouse anti‐cat CD4:FITC, Bio‐Rad, Hercules, California) and phycoerythrin‐conjugated CD8⁺ (Mouse anti‐cat CD8 alpha/beta:RPE, Bio‐Rad) for 15 minutes at 4°C in the dark. Concentration of the antibodies was 1 μg of IgG/10 μL of PBS solution. Cells were washed twice with 200 μL of flow cytometry buffer (97% PBS solution and 3% heat‐inactivated fetal bovine serum) followed by centrifugation at 1800g for 4 minutes. Cells were resuspended in flow cytometry buffer, and lymphocytes were gated for characteristic forward‐ and side‐scatter profiles. We collected 25 000 total events per sample for flow analysis. The percentage of cells stained with antibody against CD4⁺ and CD8⁺ was determined as 2‐color flow cytometry profiles (BD FACSCalibur, BD Bioscience, San Jose, California). Percentages of stained cells were calculated by the use of flow cytometer software (FCS Express 4, BD Bioscience).

For cell cycle analysis, AGS cells were treated with TIIA or DMSO as control for 48 hr. TIIA treatment concentrations were 0.625 μM, 1.25 μM, 2.5 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were harvested with trypsin/EDTA, fixed with 70% ethanol, then spun down, after which ethanol was removed. Then each sample was mixed with RNase A (100 μg/mL), incubated at 37°C for 1 hr, and stained with propidium iodide (PI) (Santa Cruz Biotechnology, Inc.) at a concentration of 100 μg/mL in the dark at room temperature for 15 min. For apoptosis analysis, AGS cells were treated with TIIA or DMSO control medium for 48 hr after 24 hr of seeding (3.5 × 10⁵ cells in 10-cm plates). TIIA treatment concentrations were 1.25 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were harvested with trypsin/EDTA, suspended, and counted. Then each sample was adjusted to a concentration of 10⁶ cells/tube and stained with Annexin V-FITC (Santa Cruz Biotechnology, Inc.) and PI (Santa Cruz Biotechnology, Inc.) dissolved in binding buffer (Santa Cruz Biotechnology, Inc.) in the dark at room temperature for 15 min. Both cell cycle distribution and apoptotic cells proportion were then analyzed with a BD FACSCanto II flow cytometer (BD Biosciences) and FCS Express 4 (BD Biosciences).

MV4-11 and MOLM13 were incubated with SYC-522 at appropriate doses (3 µM for MV4-11, 10 µM for MOLM13) for up to 6 days. On day 3 or 6, 100 nM mitoxantrone was added to the cells for 4 h. Then the drugs were washed away with PBS, and cells were incubated in fresh medium for another 12 h. The cells were fixed in 2% paraformaldehyde and permeabilized in ice cold 100% methanol, as described [20] (link). Cells were labeled with γH2AX- AlexaFluor488 (BD) and cPARP− AlexaFluor647 (BD). Data were acquired on the LSRII (BD) and analyzed with FCS Express4.