Flow cytometry

The Annexin V FITC apoptotic detection kit was applied for apoptotic cells detection according to the supplier’s instructions. SH-SY5Y cells were seeded at a density of 1 × 10⁶ cells/dish onto a 35-mm dish for 24 h. The cells were pre-incubated with 20 µM luteolin for 1 h. After treatment with 100 µM MPP⁺ for 24 h, the cells were stained with 100 µL Annexin V/7-ADD solution, and incubated at room temperature for 20 min in the dark. The number of apoptotic cells was analyzed using a flow cytometry (Millipore, Burlington, MA, USA).

As previously reported, single-cell suspensions were obtained on day 41 by density gradient centrifugation from the mouse spleen tissue suspension [45 (link)]. The cells were labeled with anti-CD3, anti-CD4, anti-CD19, anti-Foxp3, anti-FcεRI, and anti-c-KIT on the surface. Intracellular expression markers were stained with Foxp3-PerCP-Cy5.5 after fixing and permeabilizing as described in the protocol. The cells were gated and demonstrated by flow cytometry(Millipore, Billerica, MA, USA), and data were analyzed with a Guava easyCyte 6-2L system using Guava Soft 3.1.1 software.

flow cytometry was performed to detect cell apoptosis. The transfected cells were cultured on a 6-well plate until the cells covered more than 70% of the well, then apoptosis kit was used to detect apoptosis. Briefly, the cells were digested with trypsin to obtain the cell suspension, and collected with the cell supernatant in a 5ml centrifuge tube. After centrifuging for 5 min, precipitated cells were collected and centrifugated again for 3 min. Then 200 uL 1×binding buffer and 10 um Annexin V-APC staining was added, and the cell apoptosis was detected by flow cytometry (Millipore) after 15 min at room temperature in dark.

Cells were harvested with trypsin when they have grown to a coverage rate of about 80%. After centrifuging at 1300 rpm for 5 min, the cells were washed by D-Hanks (pH 7.4) precooled at 4 °C and fixed in 75% ethanol precooled at 4 °C for 2 h. Then, the cells were washed with D-Hanks once again after discarding ethanol. Finally, cells were stained by the working solution (PI: RNase stock solution: D-Hanks = 25: 10: 1000) for 30 min in the dark. The cell solution was collected for cell cycle analysis by flow cytometry (Millipore, USA).

Analysis of apoptosis on the indicated cell lines was performed by Annexin V/propidium iodide double staining followed by Flow cytometry (Guava Technologies, Millipore, USA). The cells 2 days after transfection were collected and subjected to analysis. A minimum of 5000 cells were then analyzed by FACScan with guavaSoft 3.1.1 software (Guava Technologies, Millipore, USA) for acquisition and analysis in three independent biological replicates.

The cells were harvested 48 h after the transfection and washed twice using cold PBS. Then cells were re-suspended in binding buffer (BD Biosciences). Annexin V-FITC was utilized to stain the cells which were then resuspended using binding buffer (100 μL), before 5 mL of allophycocyanin-annexin V (BD Biosciences) and 50 mg/mL propidium iodide (Invitrogen) were added to it. Subsequently the cells were mixed and incubated for 15 min in the dark at room temperature. Flow cytometry (Millipore Guava) was used to detect and quantify the apoptotic cells based on the manufacturer's instructions.