Cellquest 3

After the freezing treatment described in the section of this paper entitled ‘S. thermophilus cryopreservation’, S. thermophilus samples were thawed at 37 °C for 10 min and harvested by centrifugation (6,000 rpm, 10 min). The harvested cells were washed twice with PBS (20 mM, pH 7.4) and resuspended in an equal volume of PBS to an approximate concentration of 10⁷ CFU/mL. Ten microliters of annexin V-FITC was first added to a cell suspension and incubated at 40 °C for 20 min in the dark. Subsequently, 10 μL of PI (50 μg/mL dissolved with 20 mM PBS) was added to the mixture and incubated at 40 °C for 10 min in the dark prior to the fluorescence measurement using a FACSort flow cytometer (BD Biosciences, Mountain View, CA, USA). Fluorescence emission spectra (excitation 470 nm, emission 490 ∼ 670 nm, BW 5 nm) were recorded, and the cell apoptosis was analyzed using Cell Quest 3.0 software (Becton Dickinson, Franklin Lakes, NJ, USA).
DNA fragmentation resulting from apoptotic signaling cascades after cold stress was detected with a TUNEL staining kit purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Furthermore, caspase activation was determined by flow cytometry after the cells had been stained with activated caspases using the CaspACE assay system (FITC-VAD-FMK, Sangon Biotech Co., Ltd, Shanghai, China).

At each time point, 1 × 10⁶ cells were collected and stored in 70% ethanol. Cells were then stained with 10 μg/ml of propidium iodide in PBS containing 1 mM of EDTA and 0.2 mg/ml RNaseA. The raw flow cytometry data were analyzed using CellQuest 3.2 software (BD Biosciences).

Analysis on the effects of CBN on cell cycle distribution was performed by the method previously described (Pozarowski and Darzynkiewicz, 2004 (link)). The cells were treated with or without CBN for 24 h in 10% FBS-supplemented medium. The cells harvested, washed twice, and fixed with 80% cold ethanol overnight at −20°C. The fixed cells were pelleted, washed with ice-cold PBS, and resuspended in staining solution containing 50 μg/mL PI and 100 μg/mL RNase. After 1 h of incubation at room temperature in the dark, the fluorescence-activated cells were sorted and cellular DNA content was analyzed with a FACSCalibur^® flow cytometer (BD Biosciences, CA, USA), and data were evaluated using CellQuest 3.0.1 software (BD Biosciences, CA, USA). The DNA content of 20,000 cells in each group was analyzed and the results were demonstrated as histograms of DNA content. The distribution of cells in each phase of cell cycle was calculated using ModFit LT 2.0 program.