Syto 16

Manufactured by Thermo Fisher Scientific

Sourced in United States

SYTO 16 is a fluorescent nucleic acid stain used for detecting and quantifying DNA and RNA in various applications, including flow cytometry, fluorescence microscopy, and plate-based assays. It exhibits excitation and emission maxima at 488 nm and 497 nm, respectively.

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Lab products found in correlation

Propidium iodide, by Thermo Fisher Scientific (3 mentions) Rnase free dnase 1, by Takara Bio (2 mentions) Alexa fluor 647 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions) Celltracker red, by Thermo Fisher Scientific (2 mentions) T75 flask, by Corning (2 mentions) Pharm lyse solution, by BD (2 mentions) Facscanto 2, by BD (2 mentions) Facsdiva software, by BD (2 mentions) Lsr fortessa 2, by BD (2 mentions) Accuri c6, by BD (2 mentions) Anti cd11b pe, by BioLegend (2 mentions) Anti cd117 pe, by BioLegend (2 mentions) Anti cd45 apc, by BD (2 mentions) Anti sca 1, by BioLegend (2 mentions) Anti cd44 percp, by BioLegend (2 mentions) Anti cd29 pe, by BD (2 mentions) Anti cd31 percp, by BioLegend (2 mentions) Anti cdh5 alexa fluor 647, by BD (2 mentions) Anti pdgfrα apc, by Thermo Fisher Scientific (2 mentions) Anti cd34 apc, by BioLegend (2 mentions)

Spelling variants of this product

Syto-16 green fluorescent nucleic acid stain (16 citations)

51 protocols using syto 16

Enucleation Efficiency Determination

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Prior to the screening assay, dead cells in the culture were removed using Dead Cell Removal kit (Miltenyi Biotec) by following the manufacturer’s instruction. Ten thousand HiDEP cells in 100 μl of the complete medium per well were plated into 96-well tissue-culture flat-bottom plates. Two concentrations (0.5 μM and 10 μM) of chemical compounds dissolved in DMSO were added to the 96-well plates. Equivalent volumes of DMSO (0.005% and 0.1% in volume) were used as control for 0.5 μM and 10 μM, respectively. Cells were then cultured in 5% CO₂ at 37 °C for 4 days.
After 4 days culture, cells were stained with 0.5 μM SYTO 16 (cell permeant nucleic acid dye) and 10 nM of SYTOX Red (non-permeant nucleic acid dye) (Invitrogen). Images of 50 fields within each well were captured using Cellomics^TM ArrayScan® VTT MCS Reader (Thermo Scientific) to measure the intensity of SYTO 16, SYTOX Red, and KuO in each cell and the collected data were analyzed on ArrayScan® VTI 700 Series. Enucleation efficiency was determined by the frequency of the cells that were negative for both dyes (representing no nucleus inside the cell) divided by the total number of cells expressing KuO and negative for SYTOX Red.

Soboleva S., Kurita R., Ek F., Åkerstrand H., Silvério-Alves R., Olsson R., Nakamura Y, & Miharada K. (2021). Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines. Communications Biology, 4, 677.

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Characterizing DNA-Loaded Extracellular Vesicles

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A mixture of three different lengths of dsDNA fragments containing 0.1 ng/ml of 400‐bp fragments (Thermo Fisher, SM1631), 0.4 ng/ml of 2000‐bp fragments (Thermo Fisher, SM1701) and 1.2 ng/ml of 5000‐bp fragments (Thermo Fisher, SM1731) was stained with 6‐μM SYTO 16 for 20 min at 37°C before nFCM analysis. A 100‐μl aliquot of the EV preparation with a particle concentration of approximately 3 × 10⁸ particles/ml was treated with 0.2‐U/μl RNase‐free DNase I (Takara, 2270A), 2‐U/μl S1 nuclease (ThermoFisher, EN0321), 1 × dsDNase (ThermoFisher, EN0771) or 200‐μg/ml proteinase K (ThermoFisher, AM2548) for 30 min at 37°C. SYTO 16 (ThermoFisher, S7578) in PBS was added to obtain a final dye concentration of 6 μM. The EV sample was incubated for 20 min at 37°C prior to the nFCM analysis.

Liu H., Tian Y., Xue C., Niu Q., Chen C, & Yan X. (2022). Analysis of extracellular vesicle DNA at the single‐vesicle level by nano‐flow cytometry. Journal of Extracellular Vesicles, 11(4), e12206.

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Time-lapse Imaging of Endothelial Cells

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Time-lapse imaging was started 3 days or 4 days after the aortic sheet culture. EC nuclei were selectively labeled with fluorescent probe SYTO-16 (50 nM, Invitrogen). Time-lapse fluorescence and phase-contrast images ware obtained every three or five minutes over 36 hours, using a confocal laser scanning microscope (FV-10i Olympus) with 10 × 0.4 NA air objective lens.

Takubo N., Yura F., Naemura K., Yoshida R., Tokunaga T., Tokihiro T, & Kurihara H. (2019). Cohesive and anisotropic vascular endothelial cell motility driving angiogenic morphogenesis. Scientific Reports, 9, 9304.

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Erythroid Cell Immunophenotyping Protocol

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Staining of single cell suspensions of spleen, bone marrow and whole blood with CD71, Ter119, CD45, Syto 16 and DAPI was performed as previously described [20 (link)]. In brief, single cells from spleen and bone marrow or blood (2 μl of whole blood added to 23 μl of normal 0.9% saline) were stained for 30 minutes at 4°C with the following antibodies: Ter119-APC (0.33 μg/ml, TER-119, Biolegend); CD71-PE-Cy7 (0.1 μg/ml, RI7217, Biolegend); and CD45-Alexa Fluor 700 (0.833 μg/ml, 30-F11, Biolegend) after blocking with 1 μg Mouse FC block (2.4G2, BD Biosciences) for 10 minutes. After washing the samples were incubated for 15 minutes at room temperature in 0.5 μM Syto 16 and 0.2 μg/ml DAPI (both Invitrogen) prepared in FACS buffer (Dulbecco’s phosphate buffered saline without calcium or magnesium (D-PBS, Gibco), supplemented with 1% bovine serum albumin (Sigma-Aldrich)). Samples were washed prior to acquisition on a BD LSRII instrument. Dead cells (DAPI^high) and doublets (FSC-A vs FSC-H and SSC-H vs SSC-W) were excluded. Erythroid cells were identified as Ter119⁺ CD45^-, with erythroblasts as CD71⁺ Syto 16^high, reticulocytes as CD71⁺ Syto 16^low and mature erythrocytes as CD71^- Syto 16^neg.

Ballesteros Reviriego C., Clare S., Arends M.J., Cambridge E.L., Swiatkowska A., Caetano S., Abu-Helil B., Kane L., Harcourt K., Goulding D.A., Gleeson D., Ryder E., Doe B., White J.K., van der Weyden L., Dougan G., Adams D.J, & Speak A.O. (2019). FBXO7 sensitivity of phenotypic traits elucidated by a hypomorphic allele. PLoS ONE, 14(3), e0212481.

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Cell Migration Dynamics Analysis

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500 μM Syto16 (Invitrogen, Italy) was used to stain living-cell nuclei. Syto 16 was administered 24 h after cell seeding (30 min incubation time at room temperature in culture medium without serum). Four independent time-lapse experiments were performed in epifluorescence by using a 20× air Nikon objective, N.A. 0.45, PlanFluor and an Eclipse Ti inverted microscope (Nikon, Japan) equipped with an incubating chamber (Okolab, Italy), a CCD ORCA R2 (Hamamatzu, Japan), a mercury arc lamp, and 3 blocks of filters (UV-2E/C, B-1°, G-2E/C, Nikon, Japan). Images were acquired for 15 hours, sampling every 15 minutes. Movies were analyzed with the ImageJ manual tracking plugin to extract the coordinates of single cells as a time function. Data were analyzed by a custom-made application written in Matlab. The following parameters were measured: cell displacement (R) (distance from the origin after 15 hours), total path covered in 15 hours (S), migration step (dS) and average speed (V) (calculated for intervals of 15 minutes). Directionality and speed of each step was measured and classified in two populations: parallel (dS_∥, V_∥) and perpendicular (dS_⊥, V_⊥) steps (Fig. 6c–g). dS was considered parallel if the angle between the step and the pattern was less than 15°, while it is considered perpendicular if this angle was between 75° and 90°.

Jacchetti E., Di Rienzo C., Meucci S., Nocchi F., Beltram F, & Cecchini M. (2014). Wharton's Jelly human Mesenchymal Stem Cell contact guidance by noisy nanotopographies. Scientific Reports, 4, 3830.

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Purification and Labeling of Mature Pc-iRBCs

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Antibody-free mature Pc-iRBCs (>95% late trophozoites/schizonts) were obtained from the blood of infected RAGKO mice. Blood samples (500 μl) were resuspended in 1 ml PBS, pipetted over 5 ml of 74% Percoll (GE Healthcare) and centrifuged (2,500 x g, acceleration/break 5/0) for 30 min at RT. The top cell layers were collected and washed with supplemented RPMI 1640 medium. Purified mature Pc-iRBCs (>95% purity) were stained with 5 μM SYTO 16 (Invitrogen) or CMTPX (Life Technologies) following the manufacturer’s instructions.

Borges da Silva H., Machado de Salles É., Lima-Mauro E.F., Sardinha L.R., Álvarez J.M, & D’Império Lima M.R. (2018). CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria. PLoS ONE, 13(8), e0202522.

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3D Histological Analysis of Tissue Samples

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For three-dimensional (3D) histological analysis, approximately 10 × 10 × 10 mm³ tissue blocks were fixed in 10% formaldehyde for 2 days and then washed in phosphate-buffered saline (PBS) for 4 days at 4 °C. Next, the specimens were cut into 350-μm-thick sections by using a vibratome. The primary antibodies anti-CD31 and anti-α-SMA were used to immunolabel the tissues. To detect the immunostained structures, Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody was used in combination with Alexa Fluor 546-conjugated goat anti-mouse or anti-rat secondary antibody (1:200, Invitrogen, Waltham, MA). The nuclei were stained using either propidium iodide or SYTO 16 (Invitrogen) at room temperature for 1 h.

Lai T.Y., Chiang T.C., Lee C.Y., Kuo T.C., Wu C.H., Chen Y.I., Hu C.M., Maskey M., Tang S.C., Jeng Y.M., Tien Y.W., Lee E.Y, & Lee W.H. (2024). Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors. British Journal of Cancer, 130(7), 1096-1108.

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Spore Viability Staining Protocol

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After different treatments, the spore suspensions (OD₆₀₀ = 1.0) were double stained with 0.5 μM SYTO16 (Invitrogen, Carlsbad, CA, United States) and 15 μM propidium iodide (PI) (Invitrogen, Carlsbad, CA, United States). Suspensions were vigorously mixed and incubated in the dark for 15 min prior to analysis as described previously (Reineke et al., 2013a (link)). Both fluorescent dyes are able to stain DNA, the membrane permeant SYTO 16 acts as an indicator for spore germination, whereas the membrane impermeant PI indicates membrane damage (Mathys et al., 2009 (link)). The spores were observed by phase contrast and fluorescence microscopy (Axio Observer. A1, Carl Zeiss, Germany).

Liang D., Zhang L., Wang X., Wang P., Liao X., Wu X., Chen F, & Hu X. (2019). Building of Pressure-Assisted Ultra-High Temperature System and Its Inactivation of Bacterial Spores. Frontiers in Microbiology, 10, 1275.

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Live Imaging of Embryonic Cortical Neurons

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Time-pregnant mice were deeply anesthetized with 10% chloral hydrate. Brains of the embryos were dissected in ice-cold PBS (Invitrogen) supplemented with 0.65% glucose. The hemispheres were separated and incubated with SYTO16 (1:1000; Invitrogen) and SiR-actin (1:1000; Spirochrome) in DMEM (Invitrogen) for 20 min on ice. After transferring the hemispheres to ice-cold DMEM, the somatosensory cortex was dissected and placed into glass-bottom dishes (Matec) that were coated with poly-l-lysine (1 μg/ml) and recombinant proteins in different combinations (10 ng each) for 1 hour at 37°C. After incubation for 20 min at RT in 100 μl of culture medium [DMEM (Invitrogen) with 25% Hanks’ balanced salt solution (Invitrogen), 10% FBS, penicillin (10,000 U/ml), streptomycin (10,000 μg/ml), 0.65% d-glucose, and 0.4 mM l-glutamine] supplemented with 0.5% methyl cellulose, compartments were filled with culture medium and 0.1 mM Hepes (Invitrogen) before live cell imaging was performed. Pictures were taken using IQ3 software with multi-positions and Z-stack protocols. To reduce exposure time and laser intensity, acquisitions were done using binning 1 × 1.

Gerstmann K., Kindbeiter K., Telley L., Bozon M., Reynaud F., Théoulle E., Charoy C., Jabaudon D., Moret F, & Castellani V. (2022). A balance of noncanonical Semaphorin signaling from the cerebrospinal fluid regulates apical cell dynamics during corticogenesis. Science Advances, 8(46), eabo4552.

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Isolation and Differentiation of Hematopoietic Cells

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T cells, B cells, Mph and DCs were sorted by flow cytometry using markers TCRβ, CD4, CD8, CD11c, CD11b, CD19, CD45R, MHC II as described before (12 (link)). Purity of sorted cells was generally >98% and viability was >97% as determined by 7-AAD staining.
Erythrocytes, normoblasts, reticulocytes and red blood cells were generated using a mouse adapted protocol for the long-term ex vivo erythroid differentiation culture protocol described by Giarratana et al (17 (link)) and Konstantinidis et al (18 (link)). Briefly, low-density bone marrow cells were cultured in erythroblast growth medium (StemPro-34 with 2.6% StemPro-34 supplement; Invitrogen), 20% BIT 9500 (StemCell Technologies), 900 ng/mL ferrous sulfate, 90 ng/mL ferrous nitrate, 10⁻⁶M hydrocortisone, penicillin/streptomycin, L-glutamine), in 3 subsequent phases. For the proliferative phase (days 1–5) cells were expanded with 100 ng/mL SCF, 5 ng/mL IL-3, and 2 IU/mL human erythropoietin (Amgen). In the differentiation step (days 6–7), the cells were supplemented with only erythropoietin in fibronectin coated plates. For enucleation (day 8–9) cells were grown without cytokines. Cells were sorted based on size gating and nucleotide staining using different combinations of Syto-16, Draq5, Mitotracker Red and MitotrackerGreen (Invitrogen).

Klarquist J., Hennies C.M., Lehn M.A., Reboulet R.A., Feau S, & Janssen E.M. (2014). STING-mediated DNA sensing promotes antitumor and autoimmune responses to dying cells. Journal of immunology (Baltimore, Md. : 1950), 193(12), 6124-6134.

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