Guava easycyte 8ht flow cytometry system

Cells were seeded in 6-well plates at a density of 1.0 × 10⁵ cells/well, serum-starved for 24 h, and treated with 10 % of FBS, L-PRP, or P-PRP for 7 days. The cells were then incubated with 10 μM camptothecin (MedChem Express, Monmouth Junction, NJ, USA) for 6 h to induce apoptosis. Cell viability and apoptosis was analyzed by staining with Annexin V and PI [37 (link)]. Briefly, cultured cells were detached, centrifuged, resuspended in phosphate buffered saline (PBS; Gibco, Carlsbad, CA, USA) and stained with an Annexin V-FITC/PI staining kit (Cell Signaling Technology, Denvers, MA, USA). Data was acquired and analyzed by flow cytometry and guavaSoft (Guava easyCyte 8HT flow cytometry system, Millipore, MA, USA).

Cell density and viability were evaluated via the trypan blue dye exclusion assay using a flow cytometer (Guava^® easyCyte™ 8HT Flow Cytometry System, Millipore, Billerica, MA, USA). A 40-mW laser with a wavelength of 635 nm was used for excitation. Samples were analyzed with a particle rate of 500 particles/µL and 1000 events. Glucose and lactic acid concentrations were measured by HPLC using a Waters chromatography system (model W1515) coupled to a refractive index detector (model W2414, Waters, Milford, MA). An Aminex^® HPX 87H (Bio-Rad, Hercules, CA) column and a 5-mM sulfuric acid mobile phase were used at a flow rate of 0.6 mL/min and temperatures of 50 and 60 °C in the detector and oven respectively. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the active monoclonal antibody in the collected samples, as described elsewhere (Carrillo-Cocom et al. 2014 ).

The ploidy of C. intermedia CBS 141442 was investigated by flow cytometry of cells stained with Sytox green (Thermo Fischer Scientific). Exponentially growing cells were harvested and fixated in cold 70% ethanol. The cells were washed in 10 mM EDTA, pH 8.0, followed by treatment with RNase A (Thermo Fischer Scientific), 0.1 mg mL⁻¹ at 37 °C for 2 h. Sytox green was added to a final concentration of 1 µM and the cells were analysed with a Guava easyCyte 8HT flow cytometry System (Merck Millipore, Darmstadt, Germany). S. cerevisiae strains of different ploidy were used as references.

The cell cycle was analyzed by flow cytometry using propidium iodide (PI; Sigma). Twenty-four hours after release from synchrony, the cells were maintained for an additional 24 h in DMEM 10% FBS. Next, the cells were collected and fixed in 70% ethanol. After washing in PBS and centrifugation, the cells were suspended in the PI (50 μg/mL) solution containing DNase-free RNAse (Invitrogen) and maintained for 30 min at 37°C. The assay was performed in a Guava® easyCyte™ 8HT flow cytometry system, and the InCyte 2.2 software (Merck Millipore, Darmstadt, Germany) was used for acquisition and analysis.