Cells were collected by trypsinization after treated with silver ion medium by 5hr, 21hr, and 48hr. Immediately, cells were stained with 7-aminoactinomycin D (7-AAD; Sigma Chemical Co., St. Louis, MO) and 20nM of SYTO 13 (Molecular Probes, Eugene, OR) in PBS for 30 minutes in the dark. 7-AAD is a cell impermeable DNA binding dye that excites at 488 nm and emits in the far red, with an emission peak at approx 670 nm. The cell-permeable SYTO 13 is a green fluorescent nucleic acid dye that excites at 488 nm and emits at 509 nm. Flow cytometry was performed with an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) that can detect SYTO 13 staining by FL1 (533/30 nM) and 7-AAD by FL3 (>670 nm), registered on a log scale. A threshold of channel 400,000 on FSC-H was set to gate out light scatter and fluorescence signals caused by debris in samples. The experiment was repeated four times with non-stained control. Samples were analyzed as: live normal cells–positive stain for SYTO 13; Necrotic cells–positive stain for SYTO 13 and 7-AAD.
Syto 13
Manufactured by BD
Sourced in United States
SYTO 13 is a nucleic acid stain used for labeling and detecting DNA in biological samples. It binds to both RNA and DNA and emits fluorescence when bound to nucleic acids. The core function of SYTO 13 is to enable visualization and quantification of nucleic acids in various applications such as cell counting, flow cytometry, and DNA gel electrophoresis.
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2 protocols using syto 13
1
Cellular Apoptosis Evaluation Using Flow Cytometry
2
Bacterial Cell Viability Assay
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To determine the changes in live cells fractions during incubation in GS, bacterial cells were collected by centrifugation, washed in saline solution and stained with fluorescent markers SYTO®13 (Life Technologies Europe BV, Denmark) and propidium iodide (PI). Negative controls (dead cells), were prepared by treatment of cell suspensions with 70% ethanol before staining. Emission was recorded at 530 ± 20 nm from SYTO®13 stained cells (green fluorescence), and at 692 ± 20 nm from PI stained cells (red fluorescence) using BD FACS Jazz Cell Sorter (BD Biosciences, USA). Subpopulations of live and dead cells were estimated from the dot plot images (cytograms) based on 100,000 events per sample using the FlowJo software (version 10, Tree Star Inc. USA). The colony forming units (CFU) were determined by plating serial dilutions of cell suspensions onto MRS agar after 4 h treatment of strains L. fermentum PCC® and L. reuteri RC-14®, and after 1 h treatment of strains L. rhamnosus LGG® and L. paracasei F-19® to obtain at least 1 Log 10 CFU reduction. The MRS agar plates were incubated anaerobically at 37 °C for 48 h. Differences in Log 10 CFU reduction between the treatments and control experiments were evaluated by the paired two-tailed Student's t-test (Microsoft Office Excel 2010).
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