Facscalibur ow cytometer

DCFH-DA method was utilized in this step. Brie y, ARPE-19 cells (1 X 10 6 cells per well) were cultured with or without different amounts of LBP in six-well plates for 24 hours, before treatment with 500 µM H 2 O 2 .
After that, these cells were cultured in the presence of DCFH-DA (10 mM) for 20 min within a dark environment. After thrice washed using cold PBS, the uorescence intensity of harvested cells was determined using a FACSCalibur ow cytometer (Beckman Coulter, Brea, CA). All experimental results are shown as a percentage relative to that of the control sample.
Measuring levels of MDA, SOD, CAT and GSH-Px activities In 1.5 mL Eppendorf tubes, ARPE-19 cells (1 X 10 6 cells per well) after different treatments were coincubated with 100 µL of RIPA lysis buffer and 10% protease inhibitor for 30 minutes. After lysis and 15 minutes of centrifugation (12000 g and 4°C), the protein in the resulting cell suspension was quanti ed using the BCA kit. Finally, the intracellular activities of MDA, SOD, and levels of CAT and GSH-Px were spectrophotometrically detected using the relevant commercial kits. Notably, SOD, CAT and GPX-Px activities were denoted as units/mg protein, while MDA levels were expressed as nmol/g protein. These experimental results are shown as percentages of the control value.

The apoptosis assay was detected by FITC coupled with Annexin-V apoptosis detection kit (Beyotime, C1062). PANC-1 cells were seeded in 6-well-plates (2×10 5 cells/well). After cells adhered overnight, they were treated with different concentrations of DMAMCL or MCL, with or without gemcitabine. After 48 h treatment, cells were washed 3 times with PBS and digested with trypsin without EDTA (Gibco) in EP tubes, followed by centrifugation at 1000 g for 3 min. The cell pellets were washed twice with PBS, following by adding 100 μL 1×binding buffer to suspend the cells. For cell staining, 5 μL FITC Annexin V and 5 μL of PI were added to each tube and incubated at room temperature (keep in dark) for 15 min. Finally, each tube was supplemented with 400 μL 1×binding buffer. The samples were analyzed by FACSCalibur ow cytometer (Beckman, BD LSRFortessa ), and the data was analyzed by FlowJo 7.6.1 software (10,0,0,0).

The spleens were gained on d42 and mononuclear cells were extracted from the spleen of mice using 75µm cell strainers. Then the red blood cells in samples were cracked with Red Cell Lysis Buffer (biosharp, China), followed by washing three times. After counting, the cells were cultured in RPMI 1640 medium with 10% FBS at a concentration of 1.0×10 7 cells/well. Next, the cells were stimulated in a 5% CO2 incubator at 37 ℃ for 5 h by using Cell Activation Cocktail (without Brefeldin A) (Biolenged, USA) and Brefeldin A Solution (1000X) (Biolenged, USA) under the guidance of the manufacturer′s instructions. After twice washing, cells were stained with APC/cy7-conjugated Zombie NIR™ (Biolenged, USA) to exclude the dead cells. Regarding surface marker analysis, cells were labeled with percp/cy5.5conjugated anti-mouse CD3ε and FITC-conjugated anti-mouse CD4 + . Then cells were washed again, followed by the xing and permeabilizing operation. Regarding intracellular cytokine marker analysis, cells were stained with BV-conjugated anti-mouse IL-4 and APC-conjugated anti-mouse IFN-γ. The amount of each antibody was added according to the manufacturer′s protocols. Then the cells were washed, followed by the resuspension and staining process. Next, cells were examined by a FACSCalibur ow cytometer (BECKMAN COULTER Cyto ex S, USA) and analyzed with FlowJo10.6.