4 6 diamidino 2 phenylindole (dapi)

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DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is a fluorescent dye used to stain nucleic acids, particularly DNA. It binds strongly to adenine-thymine (A-T) rich regions in double-stranded DNA. DAPI has an emission maximum of approximately 461 nm when bound to DNA, allowing it to be used for fluorescence microscopy applications.

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Lab products found in correlation

Lsrfortessa, by BD (9 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions) Facsaria 2, by BD (8 mentions) Annexin 5 binding buffer, by BioLegend (7 mentions) Triton x 100, by Merck Group (5 mentions) Annexin 5, by BioLegend (5 mentions) Alexa fluor 568, by Thermo Fisher Scientific (5 mentions) Facsaria 3, by BD (4 mentions) Lsm 710 confocal microscope, by Zeiss (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by BD (4 mentions) Bovine serum albumin (bsa), by Merck Group (4 mentions) Lsm 700 confocal microscope, by Zeiss (4 mentions) Facsaria, by BD (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Buffer el, by Qiagen (3 mentions) Gelvatol, by Merck Group (3 mentions) Facsdiva 8, by BD (3 mentions) Trustain fcx, by BioLegend (3 mentions) Fc block, by BD (3 mentions) Fc block, by BioLegend (3 mentions)

Spelling variants of this product

4′,6-diamidino-2-phenylindole (DAPI; (28 citations) 4′,6-Diamidino-2-Phenylindole, Dilactate (DAPI) (8 citations) 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; (2 citations)

213 protocols using 4 6 diamidino 2 phenylindole (dapi)

Single-cell Immune Profiling by Flow Cytometry

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Cells (0.5–5 × 10⁶) were pre-incubated with Mouse BD Fc Block (BD Biosciences, 553142) to avoid unspecific binding to Fc receptors and stained with appropriate antibodies at 4 °C in the dark for 30 min. For EdU staining, extracellular-stained cells were permeabilized and stained using Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher, 10632), according to the manufacturer’s instructions. For DAPI cell cycle analyses, extracellular-stained cells were permeabilized and stained with 1 µg ml⁻¹ DAPI (BioLegend, 422801) in the dark for 30 min at room temperature (RT). For Ki67 stainings, extracellular-stained cells were permeabilized and stained using either FITC Mouse Anti-Ki67 Set (BD Biosciences, 556026) or PerCP-eFluor710 Mouse Anti-Ki67 (Thermo Fisher, 46-5698-80). Cell viability was assessed using LIVE/DEAD Fixable Near-IR (775) stain (Thermo Fisher, L34976) and the cell suspensions were analyzed with an LSRFortessa (BD Biosciences). Results were analyzed using FlowJo software (Tree Star). For scRNA-seq and bulk RNA-seq, lung myeloid cells were sorted using a FACSAria III (BD Biosciences). The full list of antibodies used can be found in the Supplementary Table 1.

Vanneste D., Bai Q., Hasan S., Peng W., Pirottin D., Schyns J., Maréchal P., Ruscitti C., Meunier M., Liu Z., Legrand C., Fievez L., Ginhoux F., Radermecker C., Bureau F, & Marichal T. (2023). MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation. Nature Immunology, 24(5), 827-840.

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Cytometric Analysis of Cell Death

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J774M and BM-MDSCs were treated with NC06, Z-DEVD, Necropstatin-1 (Nec-1), Ferrostatin-1 (Fer-1) for various time. The cells cells were then stained with propidium iodide (PI) and Annexin V in Annexin V-binding buffer [10 mM Hepes (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂) and analyzed in a FACS Caliber (BD Biosciences, San Diego, CA). To analyze cells from tumors, and spleens of mice, single cells were prepared from spleens and tumors as previously described (12 (link)). Cells were stained with DAPI (Biolegend) and monoclonal antibodies that are specific for CD45.2, CD8, PD-1, CD11b, and Gr1 (Biolegend). Mouse bone marrow and spleen cells were stained with CD11b- and Gr1-specific mAb. To analyze human MDSCs, peripheral blood mononuclear cells were prepared from peripheral blood samples of healthy donors and colon cancer patients, centrifuged at 3,000 RPM for 10 min to collect buffy coat. Red cells were lysed. The cells were stained with DAPI, CD11b-, CD33-, HLA-DR5-specific mAbs (Biolegend), followed by intracellular staining using Alexa Fluor 647-conjugated anti-human neutral ceramidase antibody (Santa Cruz) and the Cytofix/Cytoperm Plus intracellular staining kit (BD Biosciences). The stained cells were analyzed in a LSR Fortessa cytometer (BD Biosciences). Data were analyzed with FlowJo V10 program (Flow Jo, Ashland, OR).

Zhu H., Klement J., Lu C., Redd P.S., Yang D., Smith A.D., Poschel D.B., Zou J., Liu D., Wang P.G., Ostrov D., Coant N., Hannun Y.A., Colby A.H., Grinstaff M.W, & Liu K. (2021). Asah2 represses the p53-Hmox1 axis to protect myeloid-derived suppressor cells from ferroptosis. Journal of immunology (Baltimore, Md. : 1950), 206(6), 1395-1404.

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Flow Cytometry-based Cell Profiling

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For flow cytometry, red blood cells were lysed with EL Buffer on ice (Qiagen), with remaining cells washed and resuspended in 1× PBS with 1% BSA and stained for 15 min with the following antibodies: human CD45-APC, human CD33-PE-Cy7, murine CD45-PE, and DAPI (Biolegend). For cultured cells, MV-4–11 and MOLM-13 were washed in 1× PBS with 1% BSA and stained for 15 min with the following antibodies: CD11b-Fitc, CD38-PE, and DAPI (Biolegend). For annexin/propidium iodide staining, an annexin V apoptosis kit was used as per manufacturer instructions (BD Pharmingen). Cells were washed and submitted for flow cytometric analysis using a 3-laser LSRII (Becton Dickinson).

Ramsey H.E., Stengel K., Pino J.C., Johnston G., Childress M., Gorska A.E., Arrate P.M., Fuller L., Villaume M., Fischer M.A., Ferrell PB J.r., Roe C.E., Zou J., Lubbock A.L., Stubbs M., Zinkel S., Irish J.M., Lopez C.F., Hiebert S, & Savona M.R. (2021). Selective Inhibition of JAK1 Primes STAT5-Driven Human Leukemia Cells for ATRA-Induced Differentiation. Targeted oncology, 16(5), 663-674.

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Immunohistochemical Analysis of Alzheimer's Pathology

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Paraffin-embedded tissue was sectioned with a thickness of 6 μm. One slide per animal was used for staining, each containing 5 equally spaced sections. Cresyl violet staining was performed to visualize neurons^{22 (link)}. Antibody staining was performed as previously described^{114 (link)}, except that all primary antibodies were incubated overnight at RT, followed by another overnight at 4 °C. For Aβ staining, the Mouse On Mouse detection kit (Vector labs) was used according to manufacturer’s instructions. The following primary antibodies were used: mouse anti-human Aβ (1:150; Covance); chicken anti-GFAP (1:150; Abcam). Cy2/Cy3-conjugated anti-mouse/chicken secondary antibodies (1:150; Jackson Immunoresearch) were used. For counterstaining, 4’,6-diamidino-2-phenylindole (1:5000; Biolegend) was used.

Suzzi S., Croese T., Ravid A., Gold O., Clark A.R., Medina S., Kitsberg D., Adam M., Vernon K.A., Kohnert E., Shapira I., Malitsky S., Itkin M., Brandis A., Mehlman T., Salame T.M., Colaiuta S.P., Cahalon L., Slyper M., Greka A., Habib N, & Schwartz M. (2023). N-acetylneuraminic acid links immune exhaustion and accelerated memory deficit in diet-induced obese Alzheimer’s disease mouse model. Nature Communications, 14, 1293.

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Immunofluorescence Staining of Nasal Tissue

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The frozen sections from human nasal tissue were blocked with 10% goat serum for 30 min and stained first with IL‐19 (1:50; Abcam) and MMP‐9 antibodies (1:50; Abcam) overnight at 4°C, separately. HNECs were fixed with 4% paraformaldehyde, blocked in 1% bovine serum albumin for 30 min, and then stained first with MMP‐9 or p65 antibodies (1:500; Cell Signaling Technology) overnight at 4°C. Subsequently, the frozen tissue sections and HNECs were incubated with Alexa Fluor 594‐/or 488‐conjugated secondary antibodies (1:1000; Invitrogen) for 1 h at room temperature. Finally, the slices were stained with 4′,6‐diamidino‐2‐phenylindole (1:30,000; Biolegend) to mark the nuclei, and sealed with antifade mountant (Invitrogen). IF was examined at ×400 magnification under a microscope (Leica), or at ×630 magnification in a confocal microscope (Carl Zeiss).

Li X., Huang J., Chen X., Lai X., Huang Z., Li Y., Li S., Chang L, & Zhang G. (2021). IL‐19 induced by IL‐13/IL‐17A in the nasal epithelium of patients with chronic rhinosinusitis upregulates MMP‐9 expression via ERK/NF‐κB signaling pathway. Clinical and Translational Allergy, 11(1), e12003.

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Exosome Labeling and Cellular Uptake

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Samples of 20 μL of exosomes were mixed with 1·5 mL of Diluent C and 4 μL of the red membrane dye PKH26 (Sigma-Aldrich, St Louis, MO, USA) and incubated for 10 min. The labelled exosomes were washed twice with PBS at 100 000 g; PBS from the top of the washed labelled exosome tube was used as the control. Thereafter, 5 × 10⁵ normal peripheral blood mononuclear cells (NPBMCs) or MyLa cells were incubated with labelled exosomes (15 μL) for 24 h. Target cells that internalized the exosomes were analysed by two methods. The first method was fluorescence microscopy. Cells were centrifuged with the Shandon Cytospin^® cytocentrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA), fixed with 4% paraformaldehyde, washed with PBS, and covered with a mounting medium containing 4′,6-diamidino-2-phenylindole (BioLegend, San Diego, CA, USA). Images were taken with an Axio Imager Z2 microscope (magnitude × 40; Zeiss, Jena, Germany). The second method was fluorescence-activated cell sorting (FACS). Cells were washed with PBS and analysed for PKH26-positive cells by FACS (Gallios™; Beckman Coulter, Brea, CA, USA).

Moyal L., Arkin C., Gorovitz-Haris B., Querfeld C., Rosen S., Knaneh J., Amitay-Laish I., Prag-Naveh H., Jacob-Hirsch J, & Hodak E. (2021). Mycosis fungoides-derived exosomes promote cell motility and are enriched with microRNA-155 and microRNA-1246, and their plasma-cell-free expression may serve as a potential biomarker for disease burden. The British journal of dermatology, 185(5), 999-1012.

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Fatty Acid-Induced Adipogenesis Assay

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Appropriate concentrations of each FA for adipogenesis observed in the dose-dependent study was used in following studies. The SVC were treated by 250 μM myristic, palmitic, oleic, and linoleic acids and 100 μM stearic acid only in the commitment stage. Culture medium was refreshed every 48 h. On d 4 and 8 of adipogenic differentiation, cells were collected for mRNA extraction. On d 8, cells were also stained by 4',6-diamidino-2-phenylindole (no. 422801, BioLegend, San Diego, CA) for cell number counting using ImageJ (National Institutes of Health, https:// imagej .nih .gov/ ij/ ), and oil red O staining for lipid quantification (Yang et al., 2013) (link). Each treatment had 4 replicates.

Yanting C., Yang Q.Y., Ma G.L., Du M., Harrison J.H, & Block E. (2018). Dose- and type-dependent effects of long-chain fatty acids on adipogenesis and lipogenesis of bovine adipocytes. Journal of dairy science, 101(2).

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Semi-Automated Detection of CTCs

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To validate the semi-automated microscopic approach with the NYONE^® (SYNENTEC, Elmshorn, Germany), cultured HT29 human CRC cells (approximately 100 cells, achieved by repeated counting) were spiked into 8.2 mL of blood from healthy donors who gave written informed consent. These blood samples were then transferred into Vacutainer-CPT-tubes (BD) and processed according to the manufacturer´s guidelines. The enriched mononuclear cell (MNC)-fraction was later incubated and stained with Alexa488-conjugated anti-CD45 antibodies (#304017; Biolegend, San Diego, CA, USA) for the detection of leucocytes (green fluorescence) and Alexa647-conjugated anti-EGFR (#sc-120 AF647; SantaCruz, Dallas, TX, USA), anti-Her2 (#3244412; Biolegend), anti-EpCAM (#324212; Biolegend) and anti-pan-CK (#628604; Biolegend) antibodies against the CTC (red fluorescence). After a washing step, a buffer containing DAPI (#422801; Biolegend, San Diego, CA, USA) was added, and automated microscopy was performed using the NYONE^® cell imager using the software package YT-software (SYNENTEC, Elmshorn, Germany) (Figure 5A). A CTC was defined as being DAPI and Alexa-647-positive, as well as Alexa488-negative. A detailed protocol of the method is given in the Supplementary Methods.

Hendricks A., Brandt B., Geisen R., Dall K., Röder C., Schafmayer C., Becker T., Hinz S, & Sebens S. (2020). Isolation and Enumeration of CTC in Colorectal Cancer Patients: Introduction of a Novel Cell Imaging Approach and Comparison to Cellular and Molecular Detection Techniques. Cancers, 12(9), 2643.

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Comprehensive Murine Immune Cell Profiling

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The peripheral blood and bone marrow cells were analyzed using Aurora (Cytek) or BD FACS Aria III and the flow cytometry data was analyzed using FlowJo (Tree Star). The combinations of the following cell surface markers were used to define the peripheral blood populations: myeloid cells: CD11b⁺ or Gr-1⁺; T-cell: CD3ε⁺; B-cell: B220⁺. The following combinations of cell surface markers were used to define the bone marrow stem and progenitor cells (Lineage/Lin: CD11b, Gr-1, B220, CD3ε, Ter119): LTHSC: Lin⁻c-Kit⁺Sca1⁺Flk2⁻CD150⁺CD48⁻; MPP2: Lin⁻c-Kit⁺Sca1⁺Flk2⁻CD150⁺CD48^+; MPP3: Lin⁻c-Kit⁺Sca1⁺Flk2⁻CD150⁻CD48⁺; MPP4: Lin⁻c-Kit⁺Sca1⁺Flk2⁺CD150⁻CD48⁺; STHSC: Lin⁻c-Kit⁺Sca1⁺Flk2⁻CD150⁻CD48⁻; MEP: Lin⁻c-Kit⁺Sca1⁻CD34⁻CD16/32⁻; CMP: Lin⁻c-Kit⁺Sca1⁻CD34^midCD16/32^mid; GMP: Lin⁻c-Kit⁺Sca1⁻CD34⁺CD16/32⁺. For bone marrow stem and progenitor cell analysis, DAPI (Biolegend) was used to distinguish dead cells. Representative examples of flow cytometry gating can be found in Figure S2C. The antibodies were used at optimized dilutions listed in Table S1.

Liu S., Adams S.E., Zheng H., Ehnot J., Jung S.K., Jeffrey G., Menna T., Purton L.E., Lee H, & Kurre P. (2024). Dynamic Tracking of Native Polyclonal Hematopoiesis in Adult Mice. bioRxiv.

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Optimizing Flow Cytometry Compensation

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All experiments to establish a median mismatch index (MMI) were done using a human CD4 evaluation kit (BD Biosciences, 566352) except for the Nova Yellow 690, which was purchased from Thermo Fisher Scientific (H001T03Y05). The following fluorochromes were used: BUV615, BUV661, and BUV737 (under 355-nm excitation); BV421, BV650, and BV711 (under 405-nm excitation); FITC and BB700 (under 488-nm excitation); PE-CF594, PE-Cy5, and Nova Yellow 690 (under 561-nm excitation); and allophycocyanin and Alexa Fluor 700 (under 640-nm excitation). DAPI, CD14 PE-Cy5 (BioLegend, 301863), CD3 allophycocyanin-R700 (BD Biosciences, 565120), CD4-allophycocyanin (BD Biosciences, 566915), CD8-BV786 (BioLegend, 344740), CD19 BB700 (BD Biosciences, 566397), and CD45RA-BV711 (BioLegend, 304137) were used to generate a small panel for testing the effect of compensation beads on biological interpretation in terms of the position of these populations on the plot.

Bhowmick D., Lowe S.K, & Ratliff M.L. (2023). Side-by-Side Comparison of Compensation Beads Used in Polychromatic Flow Cytometry. ImmunoHorizons, 7(12), 819-833.

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