Typhoon 9410

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

The Typhoon 9410 is a high-performance fluorescence and luminescence imaging system designed for a variety of laboratory applications. It provides sensitive detection and quantitative analysis of fluorescent and luminescent samples.

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Lab products found in correlation

78 protocols using typhoon 9410

Telomere Length Measurement by Southern Blot

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Telomere length was measured by Southern blot previously described [26 (link)] and was conducted at RERF. In brief, genomic DNA was isolated from leukocytes using a DNA isolation kit (Qiagen) and digested with HinfI and RsaI (NEB). Digested DNA was separated on a 0.6% agarose gel by electrophoresis. The hybridization was performed with a [³²P] end-labeled oligonucleotide (TTAGGG)₄ probe, at 45°C overnight. The images were acquired by a phosphor imager (Typhoon 9410; GE Biosciences). Mean telomere length (terminal restriction fragment, TRF) was calculated at the NIA in an observer blind manner.

Lustig A., Shterev I., Geyer S., Shi A., Hu Y., Morishita Y., Nagamura H., Sasaki K., Maki M., Hayashi I., Furukawa K., Yoshida K., Kajimura J., Kyoizumi S., Kusunoki Y., Ohishi W., Nakachi K., Weng N.P, & Hayashi T. (2016). Long term effects of radiation exposure on telomere lengths of leukocytes and its associated biomarkers among atomic-bomb survivors. Oncotarget, 7(26), 38988-38998.

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Verteporfin Biodistribution in Pancreas

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Verteporfin was administered intravenously in four animals at 1 mg/kg (active
benzoporphyrin derivative sensitizer dose) immediately after imaging and animals were
sacrificed 60 minutes post-injection. Following euthanasia, the pancreas and surrounding
tissue were removed and frozen. Ex vivo, 2.5-mm thick tissue slices were
obtained using an in-house built acrylic tissue matrix, starting approximately 5 mm from
the rostral aspect of the tumor and moving in the caudal direction. Slices were placed
directly on a fluorescence scanning system (Typhoon 9410, GE Healthcare) and fluorescence
was measured using 633 nm excitation and a 685 nm long-pass filter for emission
collection. After imaging, the tissue slices were fixed in 10% buffered formalin,
processed using standard procedure, and then embedded in paraffin. After embedding,
4-micron-thick tissue slices were obtained, stained with hematoxylin and eosin
(H&E), and mounted on slides. Slides were examined using a light microscope (BX50,
Olympus America Inc., Center Valley, PA) and images of the microscope field of view were
acquired with a CCD camera (Insight 2.0 color, SPOT Imaging Solutions, Sterling Heights,
MI). One set of images was discarded due to poor quality, and the remaining three sets of
micrographs were analyzed. Fluorescence imaging data is presented in relative fluorescence
units (RFU).

Elliott J.T., Samkoe K.S., Gunn J.R., Stewart E.E., Gardner T.B., Tichauer K.M., Lee T.Y., Hoopes P.J., Pereira S.P., Hasan T, & Pogue B.W. (2015). Perfusion CT estimates photosensitizer uptake and biodistribution in a rabbit orthotopic pancreas cancer model: a pilot study. Academic radiology, 22(5), 572-579.

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RNA Oligonucleotide Purification and Quantification

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Reagents were obtained and used as received. DNA templates and primers were obtained from Integrated DNA Technologies. The RNA oligonucleotide competitors and precursor RNAs, obtained from Dharmacon, were 2′-ACE-protected (where ACE is 2′-bis(acetoxyethoxy)-methyl ether) and were deprotected according to the manufacturer’s protocol. All RNAs were desalted using PD10 Sephadex (GE Healthcare) columns by first pre-equilibrating the column with 25 ml water, then loading the RNA and eluting with 10 ml water, collecting 1 ml fractions. Autoradiography images were obtained using a Typhoon 9410 variable mode imager (GE Healthcare) and quantified using Quantity One (Bio-Rad) software. All UV-vis measurements for RNA quantification were obtained using a Beckman Coulter DU800 UV-vis spectrophotometer by heating the RNA to 90 °C and measuring the absorption at 260 nm. Complementary DNA (cDNA) samples were quantified using an Agilent Technologies 2100 Bioanalyzer (Model: G1939A) and an Invitrogen Qubit 2.0 Fluorimeter. Subsequent sequencing was carried out on a Life Technologies Ion Proton sequencer with at least 200-fold coverage. EGM-2 Bullet Kit tissue culture medium obtained from Lonza (CC-3162) was used to grow HUVECs directly. All cells were cultured at 37°C in 5% CO₂ in 100-mm-diameter dishes unless stated otherwise.

Haniff H.S., Knerr L., Liu X., Crynen G., Boström J., Abegg D., Adibekian A., Lekah E., Wang K.W., Cameron M.D., Yildirim I., Lemurell M, & Disney M.D. (2020). Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold–small molecule interactions. Nature chemistry, 12(10), 952-961.

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Transcriptional Assays on pyrG Promoter

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The transcription assays on the pyrG promoter and its variants were performed in 10 μl containing 250 nM RNAP holoenzyme, 250 nM DNA, 100 μM GTP and ³²P-labeled pGpGpG primer in the transcription buffer [40 mM Tris–HCl (pH 8 at 25°C), 30 mM KCl, 10 mM MgCl₂, 15 μM acetylated BSA and 1 mM DTT]. RNAP–DNA–primer were pre-incubated at room temperature for 10 min. After adding GTP, the samples were incubated at 37°C for 10 min and the reaction was stopped by adding 10 μl of stop buffer (90% formamide, 50 mM EDTA, xylene cyanol and bromophenol blue). The transcription assay on the pyrBI promoter was performed in 10 μl containing 250 nM RNAP holoenzyme, 250 nM DNA, 100 μM GTP, 100 μM ATP, [γ-³²P] ATP and 5 mM to 10 μM UTP. The reaction products were electrophoretically separated on a denaturing 24% polyacrylamide/7 M urea gel and visualized with a phosphorimager (Typhoon 9410, GE Healthcare).

Shin Y., Hedglin M, & Murakami K.S. (2020). Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase. Nucleic Acids Research, 48(4), 2144-2155.

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Native PAGE Analysis of NLP Samples

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Equal mass aliquots of NLP samples (1.5 μg) were diluted with 2x native gel sample buffer (Invitrogen) and loaded onto 4–12% gradient pre-made Tris-glycine gels (Invitrogen). Samples were electrophoresed for 2 hrs. at a constant 125 V. After electrophoresis, gels were incubated with SYPRO Ruby protein gel stain (Bio-Rad) for 2 hours and then de-stained using 10% Methanol, 7% Acetic acid. Following a brief wash with ddH₂O, gels were imaged using the green laser (532 nm) of a Typhoon 9410 (GE Healthcare) with a 610 nm bandpass 30 filter. Molecular weights were determined by comparing migration vs. log molecular weight of standard proteins found in the NativeMark standard (Invitrogen).

Coleman M.A., Cappuccio J.A., Blanchette C.D., Gao T., Arroyo E.S., Hinz A.K., Bourguet F.A., Segelke B., Hoeprich P.D., Huser T., Laurence T.A., Motin V.L, & Chromy B.A. (2016). Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles. PLoS ONE, 11(3), e0150166.

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Proteomic analysis of plasma and serum

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Crude and high abundant protein depleted plasma and serum samples were separated in two dimensions using the GE Life Sciences Ettan DIGE system protocol. Briefly, each sample (50 μg) was minimally labeled with 1 μl of 200 pM Cy2, Cy3 or Cy5 for 30 minutes. Labeling reactions were stopped by the addition of 1 μl of 1 mM lysine. The samples were pooled together and added to rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1.2% DeStreak, 1% pharmalytes). A final volume of 450 μl sample was loaded onto 24 cm pH 3–10NL Immobiline DryStrips (GE Life Sciences) and focused by active overnight rehydration, followed by isoelectric focusing for a total of 62,500 Vhrs. Strips were equilibrated in SDS equilibration buffer (6 M urea, 30% glycerol, 2% SDS) for 15 min with 10 mg/ml DTT, then 15 min in fresh buffer with 25 mg/ml 15 min with IAA, then applied to DIGE gels (GE Life Sciences) for 2nd dimension separation. The resulting CyDye labeled protein gels were scanned using 100 micron resolution on Typhoon 9410 (GE Life Sciences).

Haudenschild D.R., Eldridge A., Lein P.J, & Chromy B.A. (2014). High abundant protein removal from rodent blood for biomarker discovery. Biochemical and biophysical research communications, 455(0), 84-89.

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Quantifying Protein Expression via Western Blot

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Whole cell extracts were lysed in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% deoxycholate Na, 1% NP-40, 0.1% SDS, 100 mM PMSF, 1 mM pepstatin A and 1 mM E64). Protein concentration in the extracts was determined with a protein assay system (BioRad). 5-20 μg of protein was separated on 8-12% SDS polyacrylamide gel and transferred to PVDF membrane (IPFL00010, Millipore). The membranes were blocked with SuperBlock Blocking Buffer (Thermo Scientific) and then probed with antibodies specific to corresponding proteins. Membranes were treated with Alexa488-conjugated secondary antibodies (A11029, Invitrogen), band detection was performed using variable mode imager Typhoon9410 (GE Healthcare).

Dugina V., Alieva I., Khromova N., Kireev I., Gunning P.W, & Kopnin P. (2016). Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells. Oncotarget, 7(45), 72699-72715.

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Mapping Nucleosome Sliding Kinetics

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Mapping was carried out using X. laevis histone octamers with the H3(C110A) substitution to remove the naturally occurring cysteine, along with an H2B(S53C) substitution. After labeling nucleosomes with APB (41 (link)), sliding reactions were carried out in 1× slide buffer (20 mM HEPES–KOH, pH 7.6; 50 mM KCl; 5 mM MgCl₂; 0.1 mg/ml BSA; 10 mM DTT; 5% sucrose) using 150 nM nucleosome, 50 nM remodeler and 2 mM ATP. In reactions with nucleosomes containing LacO(+1) and LacO(−11) sites, LacI was added at a 5-fold molar excess over nucleosomes (750 nM), whereas for LacO(−6)-containing nucleosomes, LacI was added at a 32-fold molar excess (4.8 μM). After UV irradiation and processing (41 (link)), samples were separated on 8% polyacrylamide (19:1), 8M urea sequencing gels run at 65 W and visualized on a GE Typhoon 9410 variable mode imager. Rates of nucleosome sliding were calculated by fitting the fraction of shifted versus unshifted nucleosomes to the single-exponential function y = a*(e^−k*x) + c, where the fit parameters were a (normalized amplitude), c (constant), and k (rate constant, min⁻¹).

Nodelman I.M., Horvath K.C., Levendosky R.F., Winger J., Ren R., Patel A., Li M., Wang M.D., Roberts E, & Bowman G.D. (2016). The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome. Nucleic Acids Research, 44(16), 7580-7591.

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Musashi1 and NF-YA Protein Detection

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Cells were lysed on ice using RIPA buffer. The lysate was centrifuged at 10,000 × g for 10 min, and the protein concentration in the supernatant was determined using the microBCA protocol (Pierce, Thermo Scientific, Rockford, IL). The protein lysate was separated by SDS-PAGE and then transferred to PVDF membranes (BioRad). The membranes were blocked with 5% BSA/TBS-0.1% Tween20 and then probed with rabbit anti-Musashi1 primary antibodies (Abcam ab52865, Cambridge, MA, 1/2,000) or rabbit anti-NF-YA primary antibodies (Abcam ab6558, Cambridge, MA, 1/1,000) for 1 h at room temperature. After washing, the blots were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (SigmaAldrich, 1/50,000) and visualized by ECL plus Membrane Blotting Detection System (GE Healthcare) using a laser scanner (Typhoon 9410, GE Healthcare).

Lagadec C., Vlashi E., Frohnen P., Alhiyari Y., Chan M, & Pajonk F. (2014). The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells. Stem cells (Dayton, Ohio), 32(1), 135-144.

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Polyacrylamide Gel Electrophoresis Analysis

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The samples were analyzed on precast 15% TBE-urea polyacrylamide gels (Bio-Rad, Hercules, CA). 5 µL of sample was mixed with 5 µL of loading buffer and heated for 5 minutes at 95°C. The sample was then loaded into the gel and run for either 30 min or 50 min at 300V. The separated gels were scanned using a Typhoon 9410 variable mode imager (GE Healthcare, Piscataway, NJ). The gel images were analyzed using ImageQuant (GE Healthcare, Piscataway, NJ) to obtain lane profiles. These profiles were then curve-fit with Gaussian curves using Origin (OriginLab, Northampton, MA) to precisely determine band position and intensity.

Song Y., Liu K.J, & Wang T.H. (2014). Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture. PLoS ONE, 9(4), e94619.

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