Guava easycyte 5 flow cytometer

ES-2 and OV-90 cell lines were incubated on 6-well plates 24 h in no-FBS medium. The cells were then incubated with fucoidan (0, 25, 50, 100, 200, and 300 μg/mL) or a combination of fucoidan (300 μg/mL) with 2 μM 2-APB, 16 μM BAPTA/AM or 2 μM ruthenium red for 48 h at 37 °C and 5% CO₂. The cells were collected through 0.25% trypsin-EDTA and washed through medium before staining with 3 μM Rhod-2 and incubated at 4 °C for 30 min. Fluorescence was observed through Guava easyCyte™ 5 Flow Cytometer (Merck Millipore, Burlington, MA, USA). The experiment was repeated three times.

Yeast cells were grown in potassium- or phosphate-free YNB medium supplemented with KCl, potassium phosphate, histidine, methionine, uracil and leucine, when required. For image acquisition a Zeiss LSM 5 Exciter-AxioImager M1 confocal microscope with a Plan-Aprochromat objective (63X/1.4 Oil DIC) and Zeiss ZEN 2009 software were used. GFP was imaged with excitation at 488 nm and emission at 505–530 nm. Adobe Photoshop software was used to increase the visibility of the GFP signals and cells by linear adjustments of intensities. For flow cytometry, a Merck-Millipore Guava EasyCyte 5 Flow Cytometer was used. Fluorescence was determined after excitation at 488 nm and using the standard green 525/30 nm emission filter. For each analysis 5000 cells were used.

The cell cycle progression of ES-2 and OV-90 cells was observed through PI. The cells on 6-well plates were treated with fucoidan (0, 25, 50, 100, 200, and 300 μg/mL) for 48 h at 37 °C and 5% CO₂. The cells were rinsed with PBS. Next, cells were resuspended (1 × 10⁶ cells) and treated with RNase A (5 μL) and PI (5 μL) for 30 min at room temperature in the dark. The fluorescent intensity was calculated through Guava easyCyte™ 5 Flow Cytometer (Merck Millipore, Burlington, MA, USA). The experiment was repeated three times.

Cell surface and intracellular expression of biomarkers was investigated by flow cytometry. For cell surface analyses, the cells were kept on ice and incubated with primary antibodies for 30 min. After washings with cold PBS, the cells were incubated for further 30 min with secondary FITC-conjugated antibodies. Dead cells were labelled with the vital fluorescent dye Sytox. For intracellular analyses, the cells were permeabilised using the Fix-Perm kit from Nordic-MUbio (Susteren, The Netherlands) following the manufacturer's instructions. We measured cell-associated fluorescence using a Guava easyCyte 5 flow cytometer, controlled by GuavaSoft v.2.7 software (Merck Millipore, Billerica, MA, U.S.A.). The cytometer was equipped with 488 nm, 20 mW, blue laser light, and forward scatter (FSC) photodiode and side scatter (SSC) photomultiplier. Green fluorescence 525/30 filter, yellow 583/26 and red 680/30 filters allow analysis of fluorescence emissions from samples. Calibration of the cytometer was routinely checked using the Guava EasyCheck kit (Merck Millipore, Billerica, MA, U.S.A.) according to the manufacturer’s instructions. Raw listmode data were exported as CSV files and then imported in the software Mathematica for data processing and analyses.

Generation of ROS was observed by 2′,7′-Dichlorofluorescin diacetate (DCFH-DA, Sigma). The cells were rinsed with PBS, and stained with 10 µM DCFH-DA for 30 min at 37 °C. The cells were washed twice with PBS and incubated with diverse doses of fucoidan for 1 h at 37 °C and 5% CO₂ condition. The cells were rinsed with PBS. The fluorescent DCF intensity was observed through Guava easyCyte™ 5 Flow Cytometer (Merck Millipore, Burlington, MA, USA). The experiment was performed triplicate.

The cell viability was measured by MTT assay according to the manufacturer's protocol (Nanjing KeyGen Biotech. Co. Ltd., China). 5 × 10³ cells were plated on 96-well plates and after overnight incubation treated with the indicated drugs for 48 hours. MTT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37℃. After removal of the medium, formazan crystals were dissolved in 150 mL of DMSO. The absorbance of MTT-formazan was measured at 550 nm using a SpectraMaxM3 microplate reader (MolecularDevices, Sunnyvale, CA, USA). Apoptosis was assessed using Annexin V PE/7-AAD apoptosis assay (Nanjing KeyGen Biotech. Co. Ltd., China), stained cells were quantified using a Guava easyCyte 5 Flow Cytometer (EMD Millipore, USA).

L02 (Cat. No. GDC079) and HepG2 (Cat. No. GDC141) cells were purchased from the Chinese Academy of Sciences (Shanghai, China). In the present study, the cell deaths of two types of cells were measured using an Annexin V-FITC/PI apoptosis detection kit. First, L02 and HepG2 cells (1×10⁶ cells/ml) were plated into 6-well plates in DMEM supplemented with 10% FBS, and then treated with LSW-ET (5 ul/ml, concentration of 120.445 µg/ml), nano-LSW-low (5 ul/ml, concentration of 20.09 µg/ml), or nano-LSW-high (5 ul/ml, concentration of 120.445 µg/ml) for 24 h, respectively. Then, cells were treated with Annexin V-FITC/PI dye according to the manufacturer’s instructions, and apoptosis was assessed using a Guava easyCyte 5 Flow Cytometer (Merck, Darmstadt, Germany).