Picogreen dsdna reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, Italy

PicoGreen dsDNA reagent is a fluorescent dye used for the quantitation of double-stranded DNA (dsDNA) in solution. It binds to dsDNA and emits a strong fluorescent signal upon excitation, allowing for the sensitive and accurate measurement of dsDNA concentrations.

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PicoGreen (622 citations) Quant-iT PicoGreen dsDNA reagent (179 citations) Quant-iT PicoGreen dsDNA reagent and kits (61 citations) PicoGreen reagent (54 citations) Quant-iTTM PicoGreen® dsDNA reagent kit (6 citations) Quant-iT PicoGreen dsDNA reagents and kits (4 citations) Quanti-iT™ Picogreen® dsDNA reagent (4 citations) Quant-iT PicoGreen dsDNA reagent assay kit (4 citations) TheQuant-iT PicoGreen dsDNA reagent (2 citations) Quanti-it PicoGreen dsDNA reagent and kit (2 citations)

40 protocols using picogreen dsdna reagent

Fluorescent Labeling of Mitochondrial and Cytosolic DNA

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Fluorescent labeling of mtDNA and cytosolic DNA was conducted as described previously [33 (link)]. Cells were seeded in 24-well plates at a density of 5 × 10⁴ cells/well. 24 h later, cells were subjected to lentiviral mediated TFB2M overexpression. 48 h later, the culture medium was replaced by FBS-free growth medium containing 200 nM MitoTracker Red CMXRos (Beyotime) and was further cultured for 30 min. Then, the medium was removed, and cells were washed. 1 ml working solution of PicoGreen dsDNA Reagent (Invitrogen, P7581) was added and further incubated for 10 min at 37°C in the dark. Then, the staining solution was replaced with fresh prewarmed medium.
To detect TLR9 and mtDNA co-localization, cells with or without TFB2M overexpression were fixed with 4% paraformaldehyde, treated with 0.1% Triton X-100, and blocked with 3% BSA. Then, the cells were then incubated with anti-TLR9 (1:200, DF2970, Affinity Bioscience, Changzhou, China) at 4°C overnight. After washing, the cells were incubated with goat anti-rabbit IgG (Dylight 549-conjugated) secondary antibodies (Thermo Fisher Scientific). Then, mtDNA was detected using 1 ml working solution of PicoGreen dsDNA Reagent (Invitrogen, P7581) for 10 min at 37°C in the dark. After that, fluorescence signaling was detected using IX83 Inverted Microscope (Olympus, Tokyo, Japan).

Wu W., Zhou S., Liu T, & Liang D. (2022). Mitochondrial transcription factor B2 overexpression increases M2 macrophage infiltration via cytosolic mitochondrial DNA-stimulated Interleukin-6 secretion in ovarian cancer. Bioengineered, 13(5), 12211-12223.

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DNA Extraction and Quantification Protocol

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Total liver DNA was extracted using the Genomic-tip 20/G kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The quantification of the purified genomic DNA and PCR products was performed fluorometrically using the Picogreen ds DNA reagent (Invitrogen, Milan, Italy).

Cioffi F., Senese R., Lasala P., Ziello A., Mazzoli A., Crescenzo R., Liverini G., Lanni A., Goglia F, & Iossa S. (2017). Fructose-Rich Diet Affects Mitochondrial DNA Damage and Repair in Rats. Nutrients, 9(4), 323.

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Quantifying Mitochondrial DNA Damage

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The injured mtDNA was calculated through the ratio of long and short fragments using real-time quantitative polymerase chain reaction (RT-qPCR). (1) DNA isolation: total lung DNA was extracted by the Genomic-tip 20/G kit (Qiagen, Valencia, CA, USA). The quantification of the PCR products or purified DNA was performed fluorometrically using the Picogreen dsDNA reagent (Invitrogen, Milan, Italy). (2) RT-qPCR was performed on lung DNA extracts as previously reported (35 (link)) using the following modification: the PCR amplification was performed using the Ranger DNA Polymerase with appropriate premixes (Bioline Ltd., London, UK). The two pairs (mtDNA long fragment and short fragment) of primers (General Biosystems, Anhui, China) are shown in Table 2. (3) For amplification of the mtDNA long fragment, the standard thermocycler program included an initial denaturation at 94°C (1 min), 94°C (15 s) for 18 cycles, 65°C (12 min), and a final extension at 72°C (10 min). The short fragment of the mtDNA was amplified by the same condition, except that the extension temperature was regulated to 60°C.

Tai H., Jiang X.L., Song N., Xiao H.H., Li Y., Cheng M.J., Yin X.M., Chen Y.R., Yang G.L., Jiang X.Y., Kuang J.S., Lan Z.M, & Jia L.Q. (2021). Tanshinone IIA Combined With Cyclosporine A Alleviates Lung Apoptosis Induced by Renal Ischemia-Reperfusion in Obese Rats. Frontiers in Medicine, 8, 617393.

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Multimodal Fluorescent Labeling Protocol

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MitoTracker Red CMXRos (100 nM; CST, 9082), MitoTracker Deep Red FM (100 nM; Invitrogen, M22426), TMRE (Life Technologies, T-669), SiR-Lysosome (1 μM; Cytoskeleton inc., CY-SC012), SiR-Actin (1 μM; Cytoskeleton inc., CY-SC001), PicoGreen dsDNA Reagent (1:10000; Invitrogen, P7581), Hoechst 33342 (1:1000; Invitrogen, H3570), Hoechst 34580 (1:1000; Invitrogen, H21486), Alexa Fluor 488 Phalloidin (1:40; Invitrogen, A12379), Alexa Fluor 555 Phalloidin (1:40; Invitrogen, A34055), Alexa Fluor 568 Phalloidin (1:40; Invitrogen, A12380), CellMask Orange (1:2000; Invitrogen, C10045), JF635-HaloLigand (100 nM; gift from L. Lavis, Ashburn, VA).

Moore A.S., Coscia S.M., Simpson C.L., Ortega F.E., Wait E.C., Heddleston J.M., Nirschl J.J., Obara C.J., Guedes-Dias P., Boecker C.A., Chew T.L., Theriot J.A., Lippincott-Schwartz J, & Holzbaur E.L. (2021). Actin cables and comet tails organize mitochondrial networks in mitosis. Nature, 591(7851), 659-664.

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Quantifying Cells in Scaffold Grafts

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DNA assays were performed using PicoGreen^® dsDNA reagent (Invitrogen, Carlsbad, CA, USA). Standard curves (Supplementary Fig. 2) were created using an aliquot of the pre-seeding sample and used to determine the quantity of cells seeded on a 25 mm³ segment of scaffold. Graft samples were obtained at the ends of the graft, in the ringed segments, and in the inter-ring segments.

Best C.A., Pepper V.K., Ohst D., Bodnyk K., Heuer E., Onwuka E.A., King N., Strouse R., Grischkan J., Breuer C.K., Johnson J, & Chiang T. (2017). Designing a tissue-engineered tracheal scaffold for preclinical evaluation. International journal of pediatric otorhinolaryngology, 104, 155-160.

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Quantifying Mitochondrial DNA Levels

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DNA extracted using Genomic-tip 20/G kit (Qiagen, Valencia, CA, USA) was quantified using the PicoGreen dsDNA reagent (Invitrogen, Milan, Italy). Relative mtDNA copy numbers were measured by qPCR using iQ5 System Apparatus (Bio-Rad) and corrected by simultaneous measurement of nuclear DNA. Amplification of mitochondrial cytochrome c oxidase subunit II (COII, mitochondrial-encoded gene) and β-actin (nuclear-encoded gene) was performed using the primers listed in Table 1. The mtDNA content was calculated using ΔCt = average Ctnuclear DNA − average CtmtDNA and, then, was obtained using the formula mtDNA content = 2(2ΔCt).

Vecchione G., Grasselli E., Cioffi F., Baldini F., Oliveira P.J., Sardão V.A., Cortese K., Lanni A., Voci A., Portincasa P, & Vergani L. (2017). The Nutraceutic Silybin Counteracts Excess Lipid Accumulation and Ongoing Oxidative Stress in an In Vitro Model of Non-Alcoholic Fatty Liver Disease Progression. Frontiers in Nutrition, 4, 42.

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Quantifying Xenogeneic Dural Patch DNA

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The remnant DNA of xenogeneic dural patch was determined according to the Chinese industry standard “tissue engineered medical products—part 25: determination of DNA residues in animal tissue derived biomaterials: fluorescence staining method” (YY/T 0606.25). Before detection, the sample was dried by vacuum until it reaches constant weight which will be used for the sample dry weight in calculating remnant DNA content (ng/mg dry weight). Briefly, making DNA release from ECM materials by proteinase k digestion or/and combined with tissue homogenization, the released DNAs were subsequently purified by using nucleic acid extraction kit (4400793, 4400795, 4400675, ABI) and DNA determination using fluorescence assay with PicoGreen dsDNA reagent (P7589, Invitrogen).

Mu Y., Shao A., Shi L., Du B., Zhang Y., Luo J., Xu L, & Qu S. (2022). Immunological Risk Assessment of Xenogeneic Dural Patch by Comparing with Raw Material via GTKO Mice. BioMed Research International, 2022, 7950834.

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Quantifying Alkaline Phosphatase Activity

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ALP was investigated after 7, 14 and 21 days. To this aim, alkaline buffer solution (500 µL) and stock substrate solution (0.5 mL, 40 mg p-nitrophenyl phosphate disodium, Sigma-Aldrich, Gillingham, UK) were added to 100 µL of each lysate sample (resulting from the procedure previously reported for the PicoGreen assay), diluted in 10 mL of distilled H₂O at 37 °C for 1 h. The p-nitrophenol production was analyzed by evaluating the solution absorbance (a Leica DM2500 equipment working at 410 nm was employed). PicoGreen^® dsDNA reagent, purchased from Invitrogen (Waltham, MA, USA), was used to estimate the number of cells for each scaffold after normalization with the ALP values of absorbance.

Bellucci D., Scalzone A., Ferreira A.M., Cannillo V, & Gentile P. (2022). Adhesive Bioinspired Coating for Enhancing Glass-Ceramics Scaffolds Bioactivity. Materials, 15(22), 8080.

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Cell Viability and Proliferation Assay

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The culture medium was removed after 3 and 7 days of cell culture and the samples were transferred to new 12-well plates; subsequently, 10% PrestoBlue solution was added (5 mg/mL in DMEM; Thermo Scientific, Oxford, UK) and the multi-well plates were incubated at 37 °C for 2 h. After the supernatant removal, the solution was transferred in 96-well plates (0.2 mL) and quantified with a spectrophotometer working at 560 nm; a Filter-based FLUOstar^® Omega multi-mode reader (FLUOstar^® Omega, BMG Labtech, Ortenberg, Germany) was used. PicoGreen^® dsDNA reagent purchased from Invitrogen (Carlsbad, CA, USA) was employed to calculate the number of the cells for each sample to make a correct normalisation of the fluorescence values. The scaffolds were carefully washed with PBS after each culturing period, incubated for 3 h at 37 °C for 3 h and then frozen at −80 °C overnight in ultra-pure water (1 mL) in order to ensure cell lysis. The assay was carried on according to the protocol of the manufacturer. Fluorescence was measured at an excitation and emission wavelength of 485 and 528 nm, respectively.

Bellucci D., Scalzone A., Ferreira A.M., Cannillo V, & Gentile P. (2022). Adhesive Bioinspired Coating for Enhancing Glass-Ceramics Scaffolds Bioactivity. Materials, 15(22), 8080.

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Genomic DNA Extraction and Quantification

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The total liver DNA was extracted using the Genomic-tip 20/G kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The quantification of the purified genomic DNA and PCR products was performed fluorometrically using the PicoGreen dsDNA reagent (Invitrogen, Milan, Italy).

Cioffi F., Senese R., Petito G., Lasala P., de Lange P., Silvestri E., Lombardi A., Moreno M., Goglia F, & Lanni A. (2019). Both 3,3′,5-triiodothyronine and 3,5-diodo-L-thyronine Are Able to Repair Mitochondrial DNA Damage but by Different Mechanisms. Frontiers in Endocrinology, 10, 216.

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