Cellometer k2

Cell apoptosis was evaluated using propidium iodide (PI)/Annexin V-FITC fluorescence staining. Briefly, A549 cells were treated with specified concentrations of berberine and cinnamaldehyde alone or in combination for 48 h in the presence or absence of Z-VAD-FMK, rapamycin, or HgCL₂. 5 μl PI and 5 μl Annexin V-FITC were added to cells for 10 min, and then washed once with PBS. After staining, cells were visualized and analyzed using a Cellometer K2 (Nexcelom Bioscience).
To assess cell autophagy, A549 cells were treated with specified concentrations of berberine and cinnamaldehyde alone or in combination for 48 h under 10% serum or 2% low serum culture conditions. Cells were stained with AO (1 μg/ml) at 37°C for 30 min before observation. Red acidic vesicular organelles (AVOs) stained by AO in autophagic cells were visualized under a fluorescence microscope. Cell autophagy was further estimated via LC3-B immunofluorescent staining using PE-conjugated anti-LC3-B. Intracellular fluorescence was estimated using flow cytometry assay.

The PBMCs from rats were separated with Percoll separation medium. The gradient Percoll separation solution was prepared as follows: Percoll: 10 × Phosphate-Buffered Saline (PBS): 1 × PBS = 6.3:0.7:3. The collected peritoneal venous blood from rats was diluted with 1 × PBS in an equal proportion on a sterile ultra-clean bench. 5 mL of Percoll separation solution was added to a 15 mL centrifuge tube. The diluted blood was spread on the upper layer of the separation solution in the centrifuge tube in equal proportions and centrifuged in a horizontal centrifuge at 1,500 g for 40 min at room temperature (23 (link)). After centrifugation, the lymphocytes aggregated in the middle layer of the blood plasma and the separation solution. A Pasteur pipette was inserted into the middle layer to carefully aspirate and transfer cells to another centrifuge tube and the cells were centrifuged for 5 min at 1,500 g. The cells were washed with 5 mL of 1 × PBS and then centrifuged at 1,500 g. The supernatant was discarded, and the cells were resuspended in 1640 complete medium. The cell density was counted with a Cellometer K2 dual fluorescence cell analyzer (Nexcelom, San Diego, CA, USA), and seeded in cell culture bottles.