Picogreen

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Australia, France, Switzerland, Canada, Japan

PicoGreen is a fluorescent nucleic acid stain used for quantifying double-stranded DNA (dsDNA) in solution. It is a highly sensitive dye that binds specifically to dsDNA, enabling accurate measurement of DNA concentrations in a variety of samples.

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Lab products found in correlation

622 protocols using picogreen

Genotyping Using Affymetrix Axiom Chip

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The Genomic Core Lab quantitated all samples by Pico Green (Thermo Fisher, Pittsburgh, PA) and diluted the DNA to 23ng/ul and shipped the plates on dry ice to Affymetrix (Los Angeles, CA) for genotyping. Plates also contained randomized duplicates. Affymetrix confirmed all DNA concentrations by Pico Green assay prior to genotyping. Genotyping used a custom designed Axiom® chip (see SNP selection below), and was performed using the Affymetrix GeneTitan® system as described in the axiom user manual⁵⁴ with resultant genotype calls provided for QC and analysis.

DeMichele-Sweet M.A., Weamer E.A., Klei L., Vrana D.T., Hollingshead D.J., Seltman H.J., Sims R., Foroud T., Hernandez I., Moreno-Grau S., Tárraga L., Boada M., Ruiz A., Williams J., Mayeux R., Lopez O.L., Sibille E.L., Kamboh M.I., Devlin B, & Sweet R.A. (2017). Genetic Risk for Schizophrenia and Psychosis in Alzheimer Disease. Molecular psychiatry, 23(4), 963-972.

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Genotyping Using Affymetrix Axiom Chip

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Quantification of Neutrophil Extracellular Traps

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Bovine PMN suspended in RPMI 1640 medium were confronted with B. besnoiti tachyzoites (4 h, 37°C, 5% CO₂) at a PMN/tachyzoite ratio of 1:6 (2×10⁵ PMN:1.2×10⁶ tachyzoites, 96-well format) in the presence of non-modified ATP (P1132, Promega, USA), non-hydrolyzable ATP (ATPγS; 0.05-50 µM; 4080, Tocris, UK), or purinergic receptor antagonists (see Table 1) at a concentration range of 0.1-100 µM. After incubation, sample supernatants were analyzed for “cell-free” NETs. The remaining cells at the well bottoms were estimated for “anchored” NETs according to (44 (link)). Therefore, picogreen (Invitrogen, Eugene, USA, 1:200 dilution in 10 mM Tris base buffered with 1 mM EDTA, 50 μl/well) was added to each supernatant or pellet sample. Extracellular DNA was quantified by picogreen-derived fluorescence intensities using an automated microplate reader (Varioskan, Thermo Scientific) at 484 nm excitation/520 nm emission as described elsewhere (26 (link), 30 (link)).

Espinosa G., Conejeros I., Rojas-Barón L., Hermosilla C.R, & Taubert A. (2023). Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling. Frontiers in Immunology, 14, 1244068.

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Inhibitors Modulate Heterophil Responses

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Heterophils were pretreated with inhibitors for 30 min in a 96-well microplate and then challenged by RPMI 1640 medium (as control), FB₁ (40 μM), or zymosan (1 mg/mL, as positive control) for 1.5 h at 37°C and 5% CO₂ atmosphere. For sampling methods, a 1:200 dilution of Picogreen (Invitrogen) in 10 mM Tris-HCl buffered with 1 mM EDTA (Life Technologies Corporation) was added to each well (50 µL), 15 min after adding Picogreen solution. The plate was read by the Infiniti M200 fluorescence plate reader (Tecan, Austria) with 484 nm excitation/525 nm emission.
The inhibitor of mTOR (Rapamycin, 50 μM) was purchased from MedChemExpress. The following inhibitors of PI3K class III (3-MA, 10 μM), GLUT1 (STF-31, 1 μM), PFKFB3 enzyme (3PO, 24 mM), MCT1 (AZD3965, 1.6 μM), GLUT4 (Ritonavir, 4.2 μM), HK2 (Bromopyruvic acid, 3 mM), RAC-GTPase (NSC23766, 1.6 μM) were purchased from Sigma-Aldrich. The concentrations of used inhibitors were based on our previous studies (Jiang et al., 2021 ).

Wu H., Zhu X., Wu Z., Li P., Chen Y., Ye Y., Wang J., Zhou E, & Yang Z. (2023). The release of FB1-induced heterophil extracellular traps in chicken is dependent on autophagy and glycolysis. Poultry Science, 102(4), 102511.

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Metagenomic DNA Extraction and Sequencing

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After thawing filters to room temperature, total genomic DNA was extracted from vent fluids and seawater as previously described in Akerman and colleagues (2013). Total genomic DNA from microbial filaments on rock samples was extracted using the MoBio UltraClean Soil DNA Isolation Kit. DNA was quantified with PicoGreen (Life Technologies) on a Turner Biosystems spectrophotometer. Hypervariable region six (V6) of the 16S rRNA gene (ca. 60 nucleotides) was PCR amplified in triplicate for each sample for bacteria and archaea separately according to Eren and colleagues (2013). Genomic DNA from the rock samples was PCR amplified for bacteria only. Triplicate PCR products for each sample were pooled, cleaned using the Qiagen MinElute Kit (Qiagen, Valencia, CA), and quantified with PicoGreen (Life Technologies). Samples were then pooled and sequenced according to Meyer and colleagues (2013).

Reveillaud J., Reddington E., McDermott J., Algar C., Meyer J.L., Sylva S., Seewald J., German C.R, & Huber J.A. (2016). Subseafloor microbial communities in hydrogen‐rich vent fluids from hydrothermal systems along the Mid‐Cayman Rise. Environmental Microbiology, 18(6), 1970-1987.

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Nextera XT DNA Library Preparation

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DNA concentration was determined by PicoGreen (Life Technologies, Carlsbad, CA) fluorescence and all samples normalized to 0.2 ng/ul. One ng DNA per sample was used to produce random sequencing libraries using the Nextera XT DNA Sample Preparation Kit (Illumina Inc., San Diego, CA) that was sequenced on a MiSeq Desktop Sequencer (Illumina Inc.). The sequencing run yielded a cluster density of 541k/mm^2 (+/−24 k/mm^2) with 95.96% of clusters passing filter (+/−0.84%) for a total of DNA concentration was determined by PicoGreen (Life Technologies, Carlsbad, CA) fluorescence and all samples normalized to 0.2 ng/μl. One ng DNA per sample was used to produce random sequencing libraries using the Nextera XT DNA Sample Preparation Kit (Illumina Inc, San Diego, CA) that was sequenced on a MiSeq Desktop Sequencer (Illumina Inc). The sequencing run yielded a cluster density of 541k/mm² (+/−24k/mm²) with 95.96% of clusters passing filter (+/−0.84%) for a total of 9.95 million paired end reads. Sample representation ranged from 1.87% to 6.86% of total sequences. Similarities were determined using complete sequences (Oliviera et al., 2013) and the program Geneious 7.0.3.

Yost D.G., Tsourkas P, & Amy P.S. (2016). Experimental bacteriophage treatment of honeybees (Apis mellifera) infected with Paenibacillus larvae, the causative agent of American Foulbrood Disease. Bacteriophage, 6(1), e1122698.

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Quantitative Western Blot Analysis of PI16

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Following flow experiments, cells were lysed in SDS lysis buffer [2% SDS; 50 mM Tris pH 6.8; 10% glycerol]. Cell concentration in the lysate was quantified using PicoGreen (Invitrogen, Renfrew, UK) by mixing 100 μl of 1/1,000 cell lysate with 100 μl of 1/200 PicoGreen, both diluted in 10 mM Tris, 1 mM EDTA (pH 8). Fluorescence was measured with excitation at 485 nm and emission 520 nm and compared to a standard curve from a known cell number. 5000 cells were loaded per lane on denaturing SDS–polyacrylamide gels and blotted onto PVDF membranes. After blocking, membranes were probed with a primary rabbit anti-PI16 antibody (Sigma, UK-HPA043763; 1/3,000) overnight at 4 °C and detected with a HP-linked goat secondary antibody.

Hazell G.G., Peachey A.M., Teasdale J.E., Sala-Newby G.B., Angelini G.D., Newby A.C, & White S.J. (2016). PI16 is a shear stress and inflammation-regulated inhibitor of MMP2. Scientific Reports, 6, 39553.

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Validation of Putative Genes via Bisulfite Sequencing

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Putative genes were validated using bisulfite sequencing in a validation cohort consisting of 76 samples. DNA was quantified using Picogreen (Invitrogen, California, USA) according to the manufacturer’s protocol. Briefly, 1 μg of genomic DNA was bisulfite-converted using EZ DNA Methylation according to manufacturer’s protocol (Zymo Research, California, USA). The regions of interest were amplified by PCR using a KOD-Multi & EPi (Toyobo, Osaka Japan), purified using QIAquick PCR columns (Qiagen, Venlo, Netherlands‎), quantified using Picogreen (Invitrogen), and verified using agarose gel electrophoresis. Libraries were prepared using an Illumina TruSeq Nano DNA sample prep kit (Illumina) according to the manufacturer’s instructions and then quantified by qPCR using a CFX96 Real-Time System (Biorad, California, USA). After normalization, the prepared library was sequenced using a Miseq system (Illumina) with 300 bp paired-end reads.
Potential sequencing adapters and low-quality bases in the raw reads were trimmed using Skewer [33 (link)] and the remaining high-quality reads were mapped to the reference genome using BS-seeker2 software [34 (link)] with a 10% mis-mapping rate. To compare the CpG methylation profiles of different sample groups, only the CpG site values were selected and the Kruskal-Wallis test was performed.

Jung J., Lee Y.J., Kim C.H, & Ahn S. (2021). Landscape of epigenetically regulated lncRNAs and DNA methylation in smokers with lung adenocarcinoma. PLoS ONE, 16(3), e0247928.

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Quantification of Serum dsDNA by PicoGreen

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Serum was diluted 1:10 with digestion buffer (PBS with 10 mM MgSO₄) and stained with PicoGreen (Invitrogen), an ultrasensitive fluorescent dsDNA stain, according to the manufacturer’s instruction. Fluorescence was then measured using a Mithras (LB 940). An excess of DNase I (Invitrogen) was then added (1 U/sample) and the sample digested for 5–8 h. The intensity of PicoGreen staining (fluorescence emission) was determined every 2–3 h. To establish that digestion was complete and the DNase I was still active, the sample was spiked with 1 µg dsDNA (purified from HEK 293 cells) and the fluorescence intensity was again measured at time points 0, 5, and 12 h. By comparison with a dsDNA standard, the quantity of dsDNA present in samples was determined.

Raftery M.J., Lalwani P., Krautkrӓmer E., Peters T., Scharffetter-Kochanek K., Krüger R., Hofmann J., Seeger K., Krüger D.H, & Schönrich G. (2014). β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps. The Journal of Experimental Medicine, 211(7), 1485-1497.

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Quantifying DNA Digestion Efficiency

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Serum samples were diluted 1:50 with digestion buffer spiked with 1 μg/ml of double stranded DNA. Samples were stained with PicoGreen (Invitrogen) according to the manufacturer's instruction and incubated at 37 °C for 5 h. The reduction in PicoGreen staining (fluorescence emission, Em) was then measured using a fluorometer.

Lee Y.Y., Park H.H., Park W., Kim H., Jang J.G., Hong K.S., Lee J.Y., Seo H.S., Na D.H., Kim T.H., Choy Y.B., Ahn J.H., Lee W, & Park C.G. (2020). Long-acting nanoparticulate DNase-1 for effective suppression of SARS-CoV-2-mediated neutrophil activities and cytokine storm. Biomaterials, 267, 120389.

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