Sytox green nucleic acid stain

Manufactured by Thermo Fisher Scientific

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SYTOX Green Nucleic Acid Stain is a fluorescent dye used for the detection and quantification of DNA and RNA in a variety of biological samples. It exhibits strong fluorescence upon binding to nucleic acids, making it a useful tool for flow cytometry, fluorescence microscopy, and other analytical techniques.

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SYTOX Green (1283 citations) SYTOX Green stain (9 citations)

260 protocols using sytox green nucleic acid stain

Quantification of Cell Viability in TM7x Cells

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Pretreated TM7x cells were washed twice using saline and resuspended in saline before staining. SYTOX Green Nucleic Acid Stain (5 μM; S7020; Invitrogen) was added to each tube to stain the cells for 15 min at room temperature while avoiding light. The TM7x cells were then washed twice using saline before being fixed with 4% formaldehyde for 3 h. Images were acquired by using a Zeiss LSM 880 confocal microscope under a 63× oil immersion objective with a 488-nm excitation laser. At least three fields of view per slide and three slides per tube were acquired.
Quantitative analysis of the SYTOX fluorescence intensity was carried out at days 1, 2, and 7. Approximately 5 μM SYTOX Green Nucleic Acid Stain (S7020; Invitrogen) was added to each tube containing saline-washed TM7x cells and separated into a 96-well black flat-bottom plate with 100 μL in each well. The cells were stained for 15 min at room temperature while avoiding light. The 96-well plate was then read by a SpectraMax i3x microplate reader with SoftMax Pro software (version 7.0.3). The excitation and emission wavelengths were 488 and 523 nm. Several control wells with only saline and TM7x (free of SYTOX Green Stain) were used to deduct autofluorescence. Triton X-100 (1%; wt/vol) detergent–treated TM7x cells were used as positive controls in both the imaging and quantitative analysis procedure in Fig. 2.

Tian J., Utter D.R., Cen L., Dong P.T., Shi W., Bor B., Qin M., McLean J.S, & He X. (2022). Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment. Proceedings of the National Academy of Sciences of the United States of America, 119(2), e2114909119.

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Cell Lysis Monitoring via DNA Release Assay

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DNA release assay to monitor cell lysis upon competence induction was performed essentially as described previously (28 (link)). Sytox green nucleic acid stain (Invitrogen) fluoresces upon binding to DNA, and since the dye cannot be internalized by pneumococcal cells, emission of fluorescence is a marker for cell lysis. Briefly, cells were grown in 96-well plates (black plates, clear bottom; Corning) in the presence of 2 μM Sytox green nucleic acid stain (Invitrogen). Growth (OD₅₅₀) and fluorescence emitted (excitation and emission wavelengths, 485 and 528 nm) were measured every 5th minute using a Synergy H1 hybrid reader (BioTek). When necessary, the cultures were induced to competence at an OD₅₅₀ of ∼0.2 by the addition of 250 ng/ml CSP-1 (competence-stimulating peptide 1).

Stamsås G.A., Restelli M., Ducret A., Freton C., Garcia P.S., Håvarstein L.S., Straume D., Grangeasse C, & Kjos M. (2020). A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae. mBio, 11(5), e02461-20.

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Membrane Permeability Assay for S. aureus

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Membrane permeability assay was conducted on S. aureus (NCTC10788) using the SYTOX™ Green Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA, USA) as described previously, with some modifications.7 To obtain the fluorescence kinetics of membrane permeabilization, the bacterial suspension and peptide solutions were mixed to give a final concentration at 10 µM in a black 96-well plate, the 5 µM SYTOX Green nucleic acid stain was mixed with the reaction immediately. Then, the plate was read for 40 minutes (interval 5 minutes) directly without incubation and changes in membrane permeability were quantified via time-course analyses. All the peptides were prepared in 5% Tryptic soy broth (TSB) (v/v) in 0.85% NaCl solution (m/v) to achieve the final concentration of 10 µM. The total permeabilized cells of S. aureus were prepared by treating with 70% isopropanol and further resuspended in 5% TSB/0.85% NaCl solution after washing. The S. aureus cells resuspended in 5% TSB/0.85% NaCl solution were used as the negative control. We also included the background control with SYTOX Green dye and 5% TSB/0.85% NaCl solution only.

Liu Y., Du Q., Ma C., Xi X., Wang L., Zhou M., Burrows J.F., Chen T, & Wang H. (2019). Structure–activity relationship of an antimicrobial peptide, Phylloseptin-PHa: balance of hydrophobicity and charge determines the selectivity of bioactivities. Drug Design, Development and Therapy, 13, 447-458.

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Flow Cytometry Analysis of Chromosome Number

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Flow cytometry analyses were performed as described earlier (Murray et al. 2013 (link)). Cells were incubated until mid-log phase, and 1 mL of culture was transferred into 9 mL of ice-cold 70% ethanol and stored overnight at −20°C for fixation. Two milliliters of the fixed cells was washed with 1 mL of staining buffer (10 mM Tris-HCl at pH 7.20, 1 mM EDTA, 50 mM sodium citrate, 0.01% Triton-X-100). The cells were then harvested by centrifugation at 8000 rpm for 5 min, and the pellet was resuspended in 1 mL of staining buffer containing 0.1 mg/mL RNase A (Roche) and incubated for 30 min at room temperature. The cells were pelleted at 8000 rpm for 5 min, and the pellet was resuspended in 1 mL of staining buffer containing 0.5 μM SYTOX green nucleic acid stain (Molecular Probes). The cells were incubated in the dark for 5 min and analyzed using an Accuri C6 flow cytometer (BD Biosciences) equipped with an argon ion laser. Relative chromosome number was directly estimated from the green fluorescence (FL1-A) value of the stained cells and analyzed using BD Accuri C6 software.

Narayanan S., Janakiraman B., Kumar L, & Radhakrishnan S.K. (2015). A cell cycle-controlled redox switch regulates the topoisomerase IV activity. Genes & Development, 29(11), 1175-1187.

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Assessing Regulated Cell Death Modalities

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MCA205 or GL261 cells were seeded at 10⁵ cells/well in a 96-well plate. After incubation overnight, the cells were stained with 3.3 µM Sytox Green nucleic acid stain (molecular probes) and stimulated with 2.5 µM RAS-selective lethal 3 (RSL3) (Sigma Aldrich) for different durations (1, 3, 6 and 24 hours). Cell death was analyzed as described in Demuynck et al.36 (link) Fluorescence was measured on a Tecan Spark 20M multimode microplate reader.
The following inhibitors were used to block different cell death modalities36 37 (link): the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk, 25 µM, apoptosis, Bachem), the inhibitor of ROS and lipid peroxidation ferrostatin-1 (Fer-1, 1 µM, ferroptosis, Sigma Aldrich), the iron chelator deferoxamine (DFO, 10 µM, ferroptosis, Sigma Aldrich), the nonspecific lipophilic antioxidant α-tocopherol (α-toc, 100 µM, ferroptosis, Sigma Aldrich) and the RIPK-1 inhibitor necrostatin-1s (Nec-1s, 20 µM, necroptosis, Abcam). The cell death inhibitors were added 1 hour before RSL3 stimulation.
For recovery experiments on both MCA205 and GL261 cells, after treatment for 1, 3 or 6 hours, RSL3-conditioned medium (2.5 µM RSL3) was replaced with drug-free medium; cell death was determined 24 hours after RSL3 stimulation as described above.

Efimova I., Catanzaro E., Van der Meeren L., Turubanova V.D., Hammad H., Mishchenko T.A., Vedunova M.V., Fimognari C., Bachert C., Coppieters F., Lefever S., Skirtach A.G., Krysko O, & Krysko D.V. (2020). Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity. Journal for Immunotherapy of Cancer, 8(2), e001369.

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Fluorescence-based Tardigrade Viability Assay

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We used SYTOX green nucleic acid stain purchased from Molecular Probes (Oregon, USA) [40 ]. The SYTOX green dye is not fluorescent in aqueous solution and cannot cross either intact cell membranes or egg/embryo shells. When the cell membrane integrity is altered by cell or animal death, SYTOX green can then bind dsDNA, thereby increasing its fluorescence by a great order of magnitude (X1000) and allowing the detection of dead tardigrades by direct fluorescence at a wavelength of 523 nm. Groups of 20 tardigrades were transferred to 12-well plates. The wells were filled with 1.5 ml Chalkley’s medium. SYTOX green was added to each well on the Chalkley’s medium at a final concentration of either 0, 0.1, 1 or 10 μM, and the plates were then incubated at 15°C. Scoring of the dead tardigrades was conducted under a fluorescence microscope at either 1 h, 24 h, 48 h or 6 days as previously described for sodium azide treatments (see previous section). Each experiment was repeated three times. The animals' death scoring was immediately followed by another check by another laboratory experimenter to verify the detection of dead tardigrades.

Richaud M, & Galas S. (2018). Defining the viability of tardigrades with a molecular sensor related to death. PLoS ONE, 13(10), e0206444.

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Immune Cell Isolation and Phenotyping

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Immediately after Percoll gradient separation and washing steps, cells were stained with the following antibodies: Pacific blue-labeled mouse anti CD11b (Serotec, MCA275PB; final dilution 1:30) or Alexa647-labeled mouse anti CD45 (Serotec, MCA 43A647; final dilution 1:30), including isotype controls (IgG2a, IgG1) to control for background staining. Briefly, cells per region were incubated for 30 min at 4^∘C in antibody diluted in PBS and shielded from light. In addition, cells were incubated for 10 min with Sytox green nucleic acid stain (Molecular Probes, S7020; final dilution 1:500,000) to distinguish living from dead cells, shortly before flow cytometry. Subsequently, cells were rinsed twice in PBS, pelleted at 1200 rpm for 5 min at 4^∘C and resuspended in 300 μl PBS before they were filtered over a 70 μm size strainer to obtain a single cell suspension ready for FACS analysis and sorting.

Doorn K.J., Brevé J.J., Drukarch B., Boddeke H.W., Huitinga I., Lucassen P.J, & van Dam A.M. (2015). Brain region-specific gene expression profiles in freshly isolated rat microglia. Frontiers in Cellular Neuroscience, 9, 84.

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Allicin-Induced Membrane Permeabilization in Promastigotes

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Cell membrane permeabilization was determined using the SYTOX Green nucleic acid stain (Molecular Probes) [36 (link)] with modifications. Mid-log phase promastigotes were washed twice in HBSS and parasites (2 x 10⁶ promastigotes/mL) were incubated (15 min, 27°C) in the dark with 2 μM SYTOX Green. Cells were incubated in the presence of increasing concentrations of allicin (0, 15, 30, 60, 90 and 120 μM) for 3 h, 27°C. An aliquot of the parasite suspension (200μL/well) was transferred to a 96-well solid black microtiter plate (Costar, Corning) and fluorescence intensity was measured in a FLUOstar OPTIMA microplate reader (BMG Labtech) with excitation and emission wavelengths of 520 nm and 500 nm, respectively. Control for maximum fluorescence (100% membrane permeabilization) was obtained by the addition of 0.5% Triton X-100. Three independent experiments were carried out in triplicate.

Corral M.J., Benito-Peña E., Jiménez-Antón M.D., Cuevas L., Moreno-Bondi M.C, & Alunda J.M. (2016). Allicin Induces Calcium and Mitochondrial Dysregulation Causing Necrotic Death in Leishmania. PLoS Neglected Tropical Diseases, 10(3), e0004525.

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Multi-Labeling of Retinal Cell Types

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Single, double, and triple labeling of the retina were performed according to previously published methods [23 (link), 24 (link), 30 (link)]. Briefly, the sections were postfixed for 5 minutes in 70% ethanol, rinsed 3 × 5 minutes in 0.1 M Tris buffer and pH 7.4/0.03% Triton and blocked for 90 minutes in 10% normal donkey serum (NDS) in 0.1 M Tris buffer/0.5% Triton. Sections were then incubated with primary antibodies prepared in blocking solution overnight at room temperature. The cannabinoid-related antibodies (CB1R, NAPE-PLD, FAAH, CB2R, DAGLα, and MAGL) were also used conjointly with a known specific retinal cell-type marker (Table 1). The next day, sections were washed for 10 minutes and 2 × 5 minutes in 0.1 M Tris/0.03% Triton. Then, they were blocked in 10% NDS and 0.1 M Tris/0.5% Triton for 60 minutes and incubated with secondary antibody for one hour (Alexa 488 donkey anti-mouse and biotinylated donkey anti-rabbit followed by the addition of streptavidin-Alexa 647 (1 : 200), all prepared in blocking solution). Sections were counterstained with Sytox Green Nucleic Acid Stain (1 : 50,000; Molecular Probes, Inc., Eugene, OR), washed again in Tris buffer, and coverslipped with Fluoromount-G™ Mounting Medium (SouthernBiotech, Birmingham, AL).

Bouskila J., Javadi P., Elkrief L., Casanova C., Bouchard J.F, & Ptito M. (2016). A Comparative Analysis of the Endocannabinoid System in the Retina of Mice, Tree Shrews, and Monkeys. Neural Plasticity, 2016, 3127658.

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Immunohistochemical Analysis of Developing Limb Tissues

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Embryos were collected between embryonic days E10.5–E14. Embryos were fixed by immersion in 4% paraformaldehyde for 24 hours and forelimbs and hindlimbs were dissected. Samples were washed with PBS and cryoprotected with 30% sucrose, frozen in OCT and 12–18 μm cryosections were cut. Sections were blocked (2.5% BSA, 0.1% Triton-X100) for 1 hour and incubated with primary antibody diluted in blocking solution overnight at 4 °C. Sections were washed with PBS and incubated in the relevant secondary antibody conjugated to Alexa-Fluor 488 nm, 568 nm or 647 nm (Molecular Probes/Invitrogen) for 2 hours at RT, washed with PBS and counter-stained with DAPI (4′,6-diamidino-2-phenylindole) and/or SYTOX® green nucleic acid stain (Molecular probes/Invitrogen, ThermoFisher Scientific), prior to mounting with Fluoro-Gel mounting media (Electron Microscopy Services #17985-11).

Bosch P.J., Fuller L.C, & Weiner J.A. (2018). An essential role for the nuclear protein Akirin2 in mouse limb interdigital tissue regression. Scientific Reports, 8, 12240.

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