EMCV was labeled with Syto‐82 as described previously (Brandenburg et al, 2007 (link)). Briefly, Vero cells were infected with MOI 1 EMCV for 2 h and then incubated with 25 μM Syto‐82 (Invitrogen) at 37°C, 5% CO2 for 4 h. At end of the labeling reaction, the cells were rinsed, harvested, and freeze‐thawed twice to release the labeled virus for following experiments. Cell debris was removed by filtration through 0.45 μm filter. The viral titer was determined by plaque assay. BMDMs were treated with Syto‐82‐labeled‐EMCV at MOI 1 for 10 min, rinsed and cultured at 37°C, 5% CO2 for 30, 60, or 90 min, and subjected for live imaging analysis. To determine the co‐localization of EMCV with the endosome‐lysosome network, BMDMs were treated with Syto‐82‐labeled‐EMCV at MOI 1 for 15 min and then stained with 100 nM LysoTracker Green DND‐26 (Invitrogen) for 45 min. Syto‐82 signals were acquired on Zeiss Axio Observer 7 40X lens or 63X oil lens with ApoTome. The images were processed by using the Zeiss Zen 2012 software and quantified by ImageJ.
Syto 82
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYTO 82 is a nucleic acid stain that binds to DNA and RNA in cells. It can be used for fluorescent labeling and detection of nucleic acids in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Anti cytochrome c antibody, by BD (1 mentions)
Image it live mitochondrial transition pore assay kit, by Thermo Fisher Scientific (1 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Fluorolog 3 fluorimeter, by Horiba (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Nah13co3, by Cambridge Isotopes (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Lunascript rt supermix kit, by New England Biolabs (1 mentions)
Bovine insulin, by Merck Group (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Mscgm bulletkit, by Lonza (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
17 protocols using syto 82
1
Visualizing EMCV Infection Dynamics in Macrophages
2
Multimodal Mitochondrial Imaging Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MitoTracker® Green FM, Orange CMTMRos, and Deep Red FM, Image-IT® LIVE Mitochondrial Transition Pore Assay Kit, SYTO® 82, tetramethylrhodamine ethyl ester perchlorate (TMRE), CellEvent™ Caspase-3/7 Green Detection Reagent, JC-1 and secondary Alexa Fluor conjugated antibodies were obtained from Life Technologies (Darmstadt, Germany). The antibody against DNA was purchased from Progen Biotechnik (Heidelberg, Germany), the anti-Tom20 antibody (sc-11415) was obtained from St. Cruz Biotech (Heidelberg, Germany) and anti-cytochrome-c antibody was obtained from BD (#556432, Heidelberg, Germany). Pneumolysin (PLY) was produced and purified as previously described57 (link). 16% formaldehyde without methanol was obtained from electron microscopy science (Hatfield, PA) and CitiFluor™ CFM3 mounting medium was from Citifour (London, UK). Plasmids encoding the AT1.03YEMK fluorescence resonance energy transfer (FRET) sensors targeted to the cytosol (cytoATeam) and mitochondria (mitoATeam), respectively were kind gifts from Hiromi Imamura (Kyoto University)58 (link). pBabe-puro-IMS-RP was a gift from Peter Sorger (Addgene plasmid #24535) and mEos3.2-N1 was a gift from Michael Davidson (Addgene plasmid #54525).
3
RNA Digestion and Fluorescence Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the XRN1 digestion, the remaining RNA molecules were labeled with SYTO 82 (Life Technologies, Eugene, OR, USA) to assess the extent of digestion. SYTO type dyes show a quantum efficiency of ∼0.4 when bound to RNA and a low quantum efficiency (0.01) in the presence of mononucleotides and the buffer alone (39 (link)). The fluorescence emission spectra of labeled RNA solutions were measured from 490 to 700 nm using a Fluorolog-3 fluorimeter (Horiba Jobin Yvon, Kyoto, Japan) with 480 nm excitation. The data was analyzed using Datamax 2.0 software.
4
Quantifying Na+/K+ ATPase in Mesenteric Arteries
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rat mesenteric arteries were dissected out, mounted in a myograph and IC100 determined as described above. Arteries were then stained with 10 µM BODIPY FL Ouabain (Invitrogen, Carlsbad, CA, USA [Catalog nr. B23461]) for 15 min and SYTO-82 (diluted 1:1000, Invitrogen, Carlsbad, CA, USA [Catalog nr. S11363]) for 10 min to stain for nuclei. Using the confocal microscope a Z-stack of BODIPY FL Ouabain fluorescence intensity was obtained (excitation at 488 nm and emission at 505–530 nm). The localization to medial smooth muscle cells was verified with SYTO-16 staining (as described above). Images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA). Background fluorescence from the arterial wall without BODIPY FL Ouabain was subtracted. For negative control, arteries were first exposed to 1 mM ouabain for 30 min and then BODIPY FL Ouabain was added for another 15 min. This reduced collected fluorescence intensity by approximately 67%.
5
Paramylon Production in Euglena gracilis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Euglena gracilis NIES-48 was cultured in culture flasks (working volume: 20 mL) in a 14 h:10 h light:dark cycle with illumination approximately 150 μmol photons m−2 s−1 at 25 °C using AF-6 medium. Before cultivation with 13C-stable isotope media, Euglena gracilis cells were grown in normal AF-6 medium for at least 3 days as preculture. The cells in the preculture were transferred to AF-6 − N medium including 20 mM of NaH13CO3 (13C: 99%, Cambridge Isotope Laboratories) or NaH12CO3 (Wako Pure Chemical) for induction of 13C- or 12C-paramylon, respectively. After purging of air with filtered (0.22 μm) nitrogen gas and enclosing in a capped culture flask (working volume: 20 mL), cells were incubated in static conditions under continuous light illumination (~150 μmol photons m−2 s−1) at 28 °C for 2 days prior to cell sorting. For the evaluation of the sorting performance, the two isotope-incorporated cells were fluorescently labeled 1 h before applying them to the RIACS using different nuclear staining dyes [13C: 20 mM of Hoechst 33342 (62249, Thermo Fisher), 12C: 5 µM of SYTO 82 (S11363, Thermo Fisher)].
6
SARS-CoV-2 Detection by RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SD HotStart DNA polymerase (10 U/mL), 10 × SD polymerase reaction buffer, and MgCl2 were purchased from Bioron GmbH (Römerberg, Germany). dNTPs (10 mM each) were purchased from New England BioLabs (NEB; Ipswitch, MA, USA). Syto82 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The LunaScript® RT SuperMix Kit was purchased from New England BioLabs. Genomic DNA was purchased from Novagen. Quantitative synthetic SARS-CoV-2 RNA ORF, E, and N were purchased from ATCC. Primers were designed using the NCBI primer-BLAST program and synthesized by Integrated DNA Technology (IDT; Coralville, IA, USA). Real-time PCR reactions were carried out using the CFX96-IVD real-time PCR detection system (Bio-Rad Laboratories, Inc.).
7
Cell Culture Protocols for Cancer Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MCF7 cells were purchased from the Tissue Culture Core at Baylor College of Medicine. BT474, T47D, and THP1 cells were purchased from ATCC; Jurkat (J32) cells were a gift from Dr. Andras Heczey; hMSC cells were purchased from Lonza (PT-2501). MCF7 cells were cultured in DMEM with 10% FBS; T47D cells were cultured in RPMI with 10% FBS; BT474 cells were cultured in DMEM with 10% FBS and 15 μg/ml insulin; THP1 and Jurkat cells were cultured in RPMI with 10% FBS and 1% L-glutamine; hMSCs were cultured using the MSCGM BulletKit from Lonza. All cell lines were cultured with 1% penicillin–streptomycin (ThermoFisher Scientific) and maintained at 37 °C in a humidified incubator with 5% CO2. All cell lines were confirmed to be free of mycoplasma contamination by DNA staining with Hoechst (ThermoFisher Scientific) or Syto 82 (ThermoFisher Scientific). DMEM, RPMI, and L-glutamine were purchased from ThermoFisher Scientific, and FBS and bovine insulin were purchased from Sigma. All cell lines were authenticated by the Cell Line Authentication Testing Division of Labcorp.
8
Monitoring RV-A2 RNA Accessibility via SYTO 82
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ref. [48 (link)] was performed according to Real-Hohn et al. [49 (link)] with minor adaptations. RNA accessibility was monitored with SYTO 82 (Thermo Fisher, Waltham, MA, USA) in a Bio-Rad CFX Connect Real-Time PCR instrument (Hercules, CA, USA). Purified RV-A2 (~3.5 µg) in PBS minus PDS (control) and plus PDS at 200 µM final concentration, was incubated for 4 h at 4 °C (negligible virus breathing) or 34 °C (strong virus breathing). Unbound PDS was removed by ultrafiltration in 100 K Merck Amicon Ultra Filter units, followed by four PBS washes to eliminate the remaining unbound PDS. SYTO 82 was added to a final concentration of 5 µM, and the volumes were adjusted to 70 µL with PBS. Three 20 µL aliquots from each of these samples were dispensed into the wells of a thin-walled PCR plate, the temperature was ramped from 25–95 °C at 1.5 °C/min, and SYTO 82 light-up fluorescence was recorded. Six independent measurements were made for each condition. Data were rendered as a dot plot revealing the temperature at which the RNA becomes accessible for SYTO 82 binding [49 (link)].
9
Identifying Corrected iPSC Clones by AscI RFLPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For identification of corrected iPSC clones by AscI RFLPs, genomic DNA was first isolated using Agencourt DNAdvance Kit (A48705, Beckman Coulter Life Sciences, Indianapolis, IN, USA). PCR was then performed with Phusion® Hot Start Flex DNA Polymerase (M0535L, NEB) for 43 cycles at 60 °C in GC buffer with 4 µM SYTO-82 (S1133, Thermo Fisher Scientific). Two primers (isogenic F and R primers, Table S2 ) were used to amplify the area of interest in the RPS19 gene. DNA products were purified with Agencourt AMPure XP (A63881, Beckman Coulter Life Science). Elution volume was adjusted based on SYTO 82 relative fluorescent unit. After an overnight digestion with AscI restriction enzyme (R0558S, NEB), RFLPs were analyzed on a 1% agarose gel with ethidium bromide (54803, Lonza, Walkersville, MD, USA) in Accu GENETM 1 × Tris-acetate-EDTA buffer (50844, Lonza). AscI positive clones were then subjected to Sanger sequencing to confirm genetic correction.
10
Cytotoxicity and Phototoxicity Assessment of Nucleus Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessment of cytotoxicity and phototoxicity of PC dyes, we also used commercialized red fluorescence nucleus markers (SYTO 80, SYTO 82, SYTO 84; ThermoFisher Scientific). HeLa cells were stained with each dye in DMEM ( + ) and the time-lapse observation was performed by an inverted microscope system (IX-71; Olympus) equipped with an UPlanSApo IR 20x/0.75 objective lens (Olympus), and a CMOS camera (ORCA Flash 4.0 V3 C13440; Hamamatsu photonics). The TRITC-A-Basic fluorescent filter set (FF01-542/20, FF570-Di01, FF01-620/52; Semrock Inc.) was used for all nucleus markers. The stage incubator system (Tokai Hit Co, Ltd.) was used to keep temperature at 37 °C and 5% CO2/95% air condition. The fluorescence and bright-field time-lapse images were taken with or without excitation using an imaging software (MetaMorph; Molecular Devices) and cell proliferation rate was assessed by visual inspection from bright-field time-lapse images.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!