Draq5

Manufactured by Merck Group

Sourced in United States

DRAQ5 is a fluorescent dye that binds to DNA. It can be used to stain and visualize nucleic acids in various applications, such as flow cytometry and fluorescence microscopy.

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Lab products found in correlation

7 protocols using draq5

Collagen I Expression Quantified

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Type I collagen expression was assessed through fluorescence microscopy using an antibody against collagen I conjugated with Alexa Fluor 455. Cells were cultured with each tested compound and their mixture for 24 h, followed by fixation with 2% paraformaldehyde and permeabilization with Triton X. Subsequently, the cells were blocked with BSA 5% for 1 h and then incubated with anti-mouse antibody against collagen I for an additional 1 h at room temperature. Nuclei were marked with DRAQ5 staining (Sigma Chemical Co., St. Louis, MO, USA). Microscopic images were captured using an Olympus CKX 41 microscope (Hamburg, Germany) at an original magnification of 20×.

Filip M., Baldea I., David L., Moldovan B., Flontas G.C., Macavei S., Muntean D.M., Decea N., Tigu A.B, & Clichici S.V. (2023). Hybrid Material Based on Vaccinium myrtillus L. Extract and Gold Nanoparticles Reduces Oxidative Stress and Inflammation in Hepatic Stellate Cells Exposed to TGF-β. Biomolecules, 13(8), 1271.

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Imaging Fungal Strains with sGFP

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Microscopy experiments were performed as described previously (Bayram et al., 2019 (link)). Strains expressing sGFP were grown in Lab-Tek chambered Coverglass W/CVT (Thermo Scientific, 155360) in 400 μl GMM with required supplements for 16 h at 25 °C. DRAQ5 (Sigma) with 1:10,000 dilution was used for nuclear staining 30 min prior to imaging under microscope. Microscopic images were captured using the Olympus FV1000 confocal microscopy in 60x magnification.

Karahoda B., Pfannenstiel B.T., Sarikaya-Bayram Ö., Dong Z., Wong K.H., Fleming A.B., Keller N.P, & Bayram Ö. (2023). The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus. Fungal genetics and biology : FG & B, 169, 103836.

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Tissue Sample Enzymatic Dissociation

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Fresh samples were resected and washed twice with 1x PBS (Gibco). Each sample was cut into ~1 mm³ pieces and enzymatically digested with 10 mL digestion medium containing 1 mg/mL collagenase and 1 U/mL Dispase II (Gibco). These samples were subsequently incubated at 37 °C for 50 min and were triturated with pipette per 15 min. The suspended cells were washed with 20% fetal bovine serum (FBS) in Dulbecco’s Modified Eagle Medium (DMEM) and filtered through a 40-µm Cell-Strainer nylon mesh (BD) and centrifuged at 700 × g for 10 min. After removing the supernatant, the cell pellet was washed twice with MACS buffer (PBS containing 1% FBS, 0.5% EDTA, and 0.05% gentamycin) and then re-suspended in the sorting buffer (PBS supplemented with 1% FBS).
Subsequently, the suspended cells were stained with DRAQ5 and DAPI (Sigma) to harvest nucleated living cells. After antibody incubation, the cells were washed twice with cold PBS and reconstituted in DMEM with 20% FBS. Cell sorting was performed with a MoFlo Astrios EQ Cell Sorter (Beckman Coulter). Unstained cells were routinely used to define FACS gating parameters and sorted into DMEM media supplemented with 20% FBS.

Pu W., Shi X., Yu P., Zhang M., Liu Z., Tan L., Han P., Wang Y., Ji D., Gan H., Wei W., Lu Z., Qu N., Hu J., Hu X., Luo Z., Li H., Ji Q., Wang J., Zhang X, & Wang Y.L. (2021). Single-cell transcriptomic analysis of the tumor ecosystems underlying initiation and progression of papillary thyroid carcinoma. Nature Communications, 12, 6058.

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Multicolor Fluorescence Imaging of Actin and Nuclei

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Phalloidin—FITC 50 μg/ml (Sigma Chemical Co., St. Louis, MO, USA) a marker for the actin miofilaments (green) was used in combination with DRAQ5 (Sigma) staining (red) for nuclei. Images were recorded using a 63 times oil immersion apochromat Zeiss objective (Zeiss LSM 710 Confocal Laser Scanning unit, Carl Zeiss AG, Oberkochen, Germany). For the annexin—FITC and Phalloidin—FITC excitation/emission of 490/525 nm, for PI a 630/680 nm excitation/emission, for DRAQ5 a 646 nm excitation/detection at 681 nm was used. Image combining, processing, and analysis were performed using the standard ZEN software package (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany) [29 ].

Tudor D., Nenu I., Filip G.A., Olteanu D., Cenariu M., Tabaran F., Ion R.M., Gligor L, & Baldea I. (2017). Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy. PLoS ONE, 12(3), e0173241.

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Rhododendron Leaf Extracts Impact on Cell Cultures

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IEC6 and HaCaT cells were grown on cover glasses in 24-well plates to reach 70 % and 95 % confluence, respectively, and exposed to Rhododendron leaf extracts for 24 h as described above, while 0.5 % DMSO was used as a negative control. Cells were washed three times with PBS before fixation in 4 % PFA in 200 mM HEPES (pH 7.4) at room temperature for 20 min. After fixation, cells were washed with PBS before applying 0.2 % Triton X-100 in PBS for 5 min at room temperature, followed by several washing steps in PBS. Finally, cells were stained for 30 min at room temperature with a mixture of 3 μM FITC-labeled phalloidin (Sigma Aldrich) and 5 μM Draq5™ in PBS, the latter used as a counter-stain of nuclear DNA. Cover glasses were mounted in Mowiol for subsequent inspection by laser scanning microscopy (see below).

Rezk A., Al-Hashimi A., John W., Schepker H., Ullrich M.S, & Brix K. (2015). Assessment of cytotoxicity exerted by leaf extracts from plants of the genus Rhododendron towards epidermal keratinocytes and intestine epithelial cells. BMC Complementary and Alternative Medicine, 15, 364.

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Immunostaining of Pancreatic Tissues

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Pancreata were dissected from K8^+/+ and K8^+/− mice and frozen in O.C.T, sectioned as described above and stained using specific antibodies as described in Alam et al.20 Mouse anti‐K7 (RCK 105; Progen, Heidelberg, Germany), rat anti‐K8 (Troma I; Developmental Studies Hybridoma Bank, NIH, USA), rabbit anti‐K18 (275, kind gift from Professor J.E. Eriksson), goat anti‐insulin (polyclonal; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti‐insulin (polyclonal; Santa Cruz Biotechnology) and rabbit anti‐GLUT2 (Polyclonal; Millipore, Temecula, CA, USA) antibodies were used to stain the pancreas tissue. Cell nuclei were counterstained with DRAQ5 (Sigma‐Aldrich, Saint Louis, MI, USA). Secondary antibodies used were as follows: donkey anti‐rabbit Alexa 546, donkey anti‐goat Alexa 488, donkey anti‐rat Alexa 488 and goat anti‐mouse Alexa 488 (Molecular Probes, Eugene, OR, USA). The sections were mounted with ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Images were acquired under strict comparable conditions between genotypes and treatments using a SP5 confocal microscope (Leica, Wetzlar, Germany).

Alam C.M., Silvander J.S., Helenius T.O, & Toivola D.M. (2018). Decreased levels of keratin 8 sensitize mice to streptozotocin‐induced diabetes. Acta Physiologica (Oxford, England), 224(2), e13085.

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Multimodal Imaging of Glial Cells

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MO3.13 cells (oligodendrocytes), GL261 cells (astrocyte derived glioblastoma) or SIM A9 cells (semi adherent microglial) were plated on microscopy dishes at a density of 5×10⁵ cells per mL in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were allowed to attach overnight at 37 °C and in 5% CO₂. After cells attached, ER-Tracker™ Red (Sigma E34250) was added to the cell media at a final concentration of 1 μM along with a final concentration at 500 nM of the flavonoid dye to be tested. Cells were left to incubate for 2 hours, and then washed 5 times with PBS. Live Cell Imaging Solution (Thermo-Fisher A14291DJ) was then added, along with 10 μM of DRAQ5 (Sigma 62251) fluorescent probe, to stain cell nuclei. Cells were then left to incubate at room temperature for 30 minutes before imaging.

McDonald L., Liu B., Taraboletti A., Whiddon K., Shriver L.P., Konopka M., Liu Q, & Pang Y. (2016). Fluorescent flavonoids for endoplasmic reticulum cell imaging. Journal of materials chemistry. B, Materials for biology and medicine, 4(48), 7902-7908.

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