Coulter epics xl flow cytometer

DNA synthesis was determined by measuring the incorporation of BrdU by using a fluorescence-conjugated antibody against BrdU (BD Biosciences, San Agustin de Guadalix, Spain), co-stained with PI, and analysed in a Coulter Epics-XL flow cytometer.
To analyse the mitotic fraction, fixed cells were incubated with the anti-phospho-Histone H3 (ser 10) antibody (Merck Millipore, Madrid, Spain) followed by Cy2-conjugated secondary antibody (Jackson ImmunoResearch, Newmarket, UK). Stained cells were then counterstained with PI and analysed for Cy2 and PI fluorescence in a Coulter Epics-XL flow cytometer.

Sensory neuronal cultures were treated with cisplatin, oxaliplatin, and carboplatin at various concentrations, washed with PBS, then incubated with incubated with 10-µmol/L carboxy-H₂DCFDA (Invitrogen) in fresh PBS for 60 min to assay total ROS. Excessive probe was washed off with PBS. The cells were harvested with trypsin, washed with PBS twice, and then suspended in 500 µl PBS. The fluorescence of the labeled cells was measured by using a Coulter EPICS XL flow cytometer (Coulter) with a fluorescence excitation of 485 nm and emission at 538 nm.
To assay mitochondrial superoxide, a 1 µmol/l MitoSOX reagent working solution in HBSS/Ca/Mg buffer was made fresh. One ml of this reagent was applied added to each well of cells and the cultures incubated for 10 min at 37°C. Cultures were washed with HBSS/Ca/Mg buffer, then the cells were trypsinized and harvested. Cells were washed twice with HBSS/Ca/Mg buffer then suspended in 500 µl HBSS/Ca/Mg buffer. The fluorescence of the labeled cells was measured by using a Coulter EPICS XL flow cytometer (Coulter) with a fluorescence excitation of 510 nm and emission at 580 nm. An average of 10,000 cells from each sample was counted, and each experiment was done at least in triplicate.

Reagents were purchased from Jingmei Bioengineering Co., Ltd. and tested using Coulter Epics XL flow cytometer (Beckman Coulter, USA). 40 μL of EDTA-K2 anticoagulated blood and 5 μL of CD4-PE and CD25-FITC antibodies were added. In the same type of control tube, another 40 μL of blood was added to 5 μL of IgG1-PE and 5 μL of IgG2a-FITC, mixed well, and incubated in the dark at room temperature for 20 min. After hemolysis, add 3 mL of PBS buffer, centrifuge at 1800 r/min for 5 min, and discard the supernatant. Then, add 500 μL of PBS buffer solution and check by flow cytometry. The results are expressed as a percentage.

The KYSE150 cells were seeded at a density of 1 × 10³ per well in 96-well plates. At 12, 24, 36, or 48 h post-transfection, cells were harvested and cell proliferation was analyzed using crystal violet assay, according to the manufacturer’s instructions. For cell apoptosis analysis, cells were treated according to the instructions provided with the Hochest33342/PI apoptosis detection kit (Solarbio, China). And the apoptotic ratios were analyzed by a Coulter Epics XL flow cytometer (Beckman, USA). For colony formation assay, 1 × 10⁴ cells suspended in 0.7% soft agar were plated on the surface of 1.2% soft agar in six-well cell culture plate. After 2-weeks growth, colony was counted under ×100 microscope.

MSCs from human dental pulp were expanded as described previously [8 (link)] and transduced with lentiviral vectors pWP-green fluorescent protein (GFP-MSCs) or pWP-HIF-GFP (HIF-MSCs) as previously described [7 (link)]. Transduction efficiency was evaluated by flow cytometry (Coulter EPICS XL flow cytometer; Beckman Coulter) to determine the percentage of GFP-positive cells. The percentages of infection obtained were normally around 90%. Human dental pulp MSCs (n = 3; Inbiobank) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Sigma-Aldrich, Spain) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, Life Technologies, Thermo Fisher Scientific). Cells were grown until confluence and subsequently subcultured up to 12 passages. For stimulation assays, 5 × 10⁴ MSCs were seeded in six-well flat-bottom culture plates and, after 3 days, media were replenished and supplemented or not with 10 ng/ml recombinant human interferon (rhIFN)-gamma (Invitrogen). After 15 h, cells were lysed for subsequent analysis by quantitative polymerase chain reaction (qPCR).

Cell viability and apoptosis were analysed by flow cytometry (FCM) in a Coulter Epics XL Flow Cytometer (Beckman Coulter, Hialeah, FL-USA), using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA-USA) as recommended by the manufacturer. Briefly, cells were harvested and washed with PBS, pH 7.2. Cells were resuspended in diluted binding buffer provided with the kit (1∶10 in distilled water) at 1×10⁶ cells/ml. Five microliters FITC-Annexin V and 5 μl propidium iodide (PI; Sigma-Aldrich; St. Luis, MO-USA) were used to stain 100 μl cell suspension for 15 min at room temperature in the dark, after which each sample was diluted in 400 μl binding buffer. At least 10,000 events were analysed for each sample and percentages were calculated from the number of cells in each quadrant divided by the total number of cells.