Aqua fluorescent reactive dye

Manufactured by Thermo Fisher Scientific

Sourced in United States

Aqua fluorescent reactive dye is a laboratory reagent used to label and detect proteins, nucleic acids, and other biomolecules. It emits a bright blue-green fluorescence upon excitation, allowing for sensitive detection and quantification of labeled molecules in various applications such as gel electrophoresis, Western blotting, and flow cytometry.

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Lab products found in correlation

Lsrfortessa, by BD (3 mentions) Golgiplug, by BD (2 mentions) Fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions) Violet fluorescent reactive dye, by Thermo Fisher Scientific (2 mentions) X20 analyzer, by BD (2 mentions) Lsrii fortessa, by BD (2 mentions) Lsr 2, by BD (2 mentions) Facscanto 2, by BD (2 mentions) Fortessa, by BD (1 mentions) Apc anti human cd206, by BioLegend (1 mentions) Cytofix cytoperm, by BD (1 mentions) Pe mouse anti human cd47, by BD (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Ic fixation buffer, by Thermo Fisher Scientific (1 mentions) Facsdiva software, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Facscanto 2 cytometer, by BD (1 mentions) Normal mouse serum, by Thermo Fisher Scientific (1 mentions) Igg isotype control, by BioLegend (1 mentions) Pe cy7 anti human cd38, by BioLegend (1 mentions)

Spelling variants of this product

Fluorescent dyes (25 citations) Aqua dye (13 citations) LIVE/DEAD Aqua fluorescent reactive dye (7 citations) Live/Dead fixable aqua fluorescent reactive dye (3 citations) Aqua Fluorescent Reactive (2 citations) Aqua green-fluorescent reactive dye (2 citations) LIVE/DEAD fixable dead cell stain kit with aqua fluorescent reactive dye (2 citations)

17 protocols using aqua fluorescent reactive dye

Intracellular TNFα Quantification

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Cells were seeded into non-treated tissue culture 24-well plates or round-bottom 96-well plates. The next day cells were stimulated with the indicated TLR ligands. To measure TNFα production, BrefeldinA (BD GolgiPlug, BD Biosciences) was added to cells 30 min after stimulation, and cells were collected after an additional 5.5 h. Dead cells were excluded using a fixable live/dead stain (Violet fluorescent reactive dye, Invitrogen). Cells were stained for intracellular TNFα with a Fixation & Permeabilization kit according to manufacturer’s instructions (eBioscience).
For flow cytometry on mouse cells, dead cells were excluded using a fixable live/dead stain (Aqua fluorescent reactive dye, Invitrogen) or DAPI and all stains were carried out in PBS containing 1% BSA (w/v) and 0.1% Azide (w/v) including anti-CD16/32 blocking antibody. Cells were stained for 20 min at 4°C with surface antibodies. Data were acquired on a LSRFortessa or X20 analyzer (BD Biosciences). See Extended Data Fig 10 for gating strategies.

Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.

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Intracellular TNFα Quantification

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Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.

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Phenotyping M1, M2, and M0 Macrophages

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Flow cytometric analysis of M1-, M2-, and M0-MΦs before and after infection with Mtb was done per published procedures. MΦs were stained and analyzed on the Fortessa flow cytometer (Beckton Dickinson), and the data were processed using FlowJo v10 software (Tree Star, Inc.). Fluorochrome-conjugated antibodies used for flow cytometry were as follows: PE-Cy7 anti-human CD68 (BD Biosciences, cat no. 565595; clone: Y1/82A), PE anti-human CD14 (Invitrogen, cat no. 12-0149-42; clone: 61D3), APC anti-human CD206 (BioLegend, cat no. 321110; clone: 15-2), AF700 anti-human CD80 (BD Biosciences, cat no. 561133; clone: L307.4). Dead cells were excluded by using aqua fluorescent reactive dye (Invitrogen, cat no. L34957). GraphPad PRISM used for data analysis. Other antibodies are listed in Supplementary Tables.

Khan A., Zhang K., Singh V.K., Mishra A., Kachroo P., Bing T., Won J.H., Mani A., Papanna R., Mann L.K., Ledezma-Campos E., Aguillon-Duran G., Canaday D.H., David S.A., Restrepo B.I., Viet N.N., Phan H., Graviss E.A., Musser J.M., Kaushal D., Gauduin M.C, & Jagannath C. (2022). Human M1 macrophages express unique innate immune response genes after mycobacterial infection to defend against tuberculosis. Communications Biology, 5, 480.

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Multiparameter Flow Cytometry Protocol

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All antibodies were purchased from Biolegend (San Diego, CA, USA). Aqua fluorescent reactive dye was purchased from Invitrogen. 2x10⁶ cells were stained for surface or intracellular proteins by incubating cells with antibodies for 45 minutes on ice, fixed and permeabilized with 1x Cytofix/CytoPerm (BD Biosciences), then stained for 45 minutes for intracellular proteins with antibodies diluted in 1x Permwash, before being fixed in 2% PFA for 20 minutes as described previously (35 (link)–39 (link)). All samples were acquired on a LSRII Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo V9 software.

Sullivan P.M., Reed S.J., Kalia V, & Sarkar S. (2021). Solid Tumor Microenvironment Can Harbor and Support Functional Properties of Memory T Cells. Frontiers in Immunology, 12, 706150.

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HTLV-1 Effect on CD47 Expression

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Effector cells (729.6 uninfected, 729.6 HTLV-1_WT, and 729.6 HTLV-1_p12KO) were washed with PBS. 10⁶ cells were stained with 10 μl of PE Mouse anti-human CD47 (BD Pharmingen, Franklin Lakes, NJ) together with a viability dye (Aqua fluorescent reactive dye, Invitrogen) for 30 min at room temperature in the dark. Cells were then collected and samples analyzed by flow cytometry (BD LSR II). Data were analyzed using FlowJo Version 10.6.

Moles R., Sarkis S., Galli V., Omsland M., Artesi M., Bissa M., McKinnon K., Brown S., Hahaut V., Washington-Parks R., Welsh J., Venzon D.J., Gutowska A., Doster M.N., Breed M.W., Killoran K.E., Kramer J., Jones J., Moniuszko M., Van den Broeke A., Pise-Masison C.A, & Franchini G. (2022). NK cells and monocytes modulate primary HTLV-1 infection. PLoS Pathogens, 18(4), e1010416.

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Murine splenocyte activation and signaling

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Single-cell suspensions of murine splenocytes were stained with Aqua Fluorescent Reactive Dye (1:500, catalog no. L34966; Invitrogen), washed twice with PBS, and resuspended in culture RPMI. Cell number was adjusted to 5 × 10⁶ cells/ml in medium and plated to 24-well plates. Cells were stimulated with PMA (40 nM) for 15 min at 37°C or with SIINFEKL peptide (0.4 μg/ml) for 5 min at 37°C. Cells were then fixed by addition of IC Fixation Buffer (Invitrogen) at room temperature in the dark for 1 h and permeabilized in 1 ml of ice-cold methanol for 30 min at 4°C. Cells were washed, then stained for 1 h at room temperature with anti-CD4, anti-CD45.2, anti-pErk1/2, anti-CD8, and anti-CD45.1 antibodies. Cells were washed twice with staining buffer and analyzed using the BD LSR II flow cytometer (BD Biosciences).

Lewis E.L., Xu R., Beltra J.C., Ngiow S.F., Cohen J., Telange R., Crane A., Sawinski D., Wherry E.J, & Porrett P.M. (2021). NFAT-dependent and -independent exhaustion circuits program maternal CD8 T cell hypofunction in pregnancy. The Journal of Experimental Medicine, 219(1), e20201599.

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Flow Cytometric Immunophenotyping of CLL

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Cells were washed in PBS and stained for 20 minutes with conjugated antibodies or isotype control obtained from BioLegend, eBioscience, or BD. The following purified mouse anti-human antibodies were used for staining: anti-CD45-Pacific Blue, anti-CD45-BV605, anti-CD19-PE-Cy7, anti-CD19-APC-Cy7, CD10-BV605, anti-CD27-PE-Cy7, anti-IgD-PE, anti-ROR1-APC (clone 2A2), anti-CD5-PerCP-Cy5.5, anti-CD38-BV711, anti-CD23-FITC, anti-NKp46-FITC, anti-CD3-Pacific Blue, anti-CD4-APC-Cy7, and anti-CD8-PE. Live and dead cell staining was performed with aqua fluorescent reactive dye from Invitrogen. Peripheral blood mononuclear cells (PBMCs) from heathy donors and CLL patients were kindly provided by Dr. Vania Coelho, University College London. Flow cytometry analysis was done using a BD LSRII Fortessa using FACSDiva software (BD Biosciences), and data were analyzed with FlowJo (Tree Star).

, & Leung C.S. (2018). Analysis of ROR1 Protein Expression in Mice with Reconstituted Human Immune System Components. Journal of Immunology Research, 2018, 2480931.

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Flow Cytometry Analysis of Human Kidney Tissues

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Flow cytometry was performed on ∼2-3 g of kidney tissue from either the renal cortex or medulla as described in our previous study (Zimmerman et al., 2019c (link)). The single-cell suspension was processed similar to the mouse kidney tissues described above. Cells were incubated in 1% BSA containing the following antibodies (Table S2): eFluor450 mouse anti-human CD45, BV650 mouse anti-human HLA-DR1, BV786 mouse anti-human CD206 and aqua fluorescent reactive dye (Invitrogen, L34957). Cells were washed with 1× PBS and re-suspended in FACS buffer. All samples were analyzed on the LSRII flow cytometer (BD Biosciences) and FlowJo version 10.0 software.

Li Z., Zimmerman K.A., Cherakara S., Chumley P.H., Collawn J.F., Wang J., Haycraft C.J., Song C.J., Chacana T., Andersen R.S., Croyle M.J., Aloria E.J., Hombal R.P., Thomas I.N., Chweih H., Simanyi K.L., George J.F., Parant J.M., Mrug M, & Yoder B.K. (2023). A kidney resident macrophage subset is a candidate biomarker for renal cystic disease in preclinical models. Disease Models & Mechanisms, 16(1), dmm049810.

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Quantifying HLA Surface Expression

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HLA surface expression was determined using the QIFIKIT bead-based quantification flow cytometric assay (Dako, catalog no. K007811–8) according to the manufacturer's instructions. In brief, cells were stained either with the pan-HLA class I-specific W6/32 mAb, the HLA-DR–specific L243 mAb (produced in-house), or IgG isotype control (BioLegend, catalog no. 400202, RRID: AB_2927399, clone MOPC-173), respectively. Polyclonal goat FITC anti-mouse antibody (Dako, catalog no. F047902, RRID: AB_578665) was used as a secondary antibody. After washing with normal mouse serum (eBioscience, catalog no. 24–5544–94) surface marker staining was performed using PE/Cy7 anti-human CD38 (BioLegend, catalog no. 356608, RRID: AB_2561903, clone HB-7), APC anti-human CD34 (BD Biosciences, catalog no. 555824, RRID: AB_398614, clone 581), and Pacific Blue anti-human CD45 (BD Biosciences, catalog no. 642275, RRID: AB_1645755, clone 2D1) antibodies. Aqua fluorescent reactive dye (Invitrogen, catalog no. L34957) was used as viability marker. Analyses were performed on a FACS Canto II cytometer (BD Biosciences). Only cell populations with ≥100 cells were analyzed for their HLA surface expression.

Nelde A., Schuster H., Heitmann J.S., Bauer J., Maringer Y., Zwick M., Volkmer J.P., Chen J.Y., Stanger A.M., Lehmann A., Appiah B., Märklin M., Rücker-Braun E., Salih H.R., Roerden M., Schroeder S.M., Häring M.F., Schlosser A., Schetelig J., Schmitz M., Boerries M., Köhler N., Lengerke C., Majeti R., Weissman I.L., Rammensee H.G, & Walz J.S. (2023). Immune Surveillance of Acute Myeloid Leukemia Is Mediated by HLA-Presented Antigens on Leukemia Progenitor Cells. Blood Cancer Discovery, 4(6), 468-489.

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Vaccine-Induced Immune Response in Mice

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Healthy 6–8 week old mice (n = 5 per group) were vaccinated subcutaneously (0.5 μg per antigenic peptide, 30 μg PC7A nanoparticle) three times in five day intervals. Twenty four hours after the final vaccination, mice were sacrificed and inguinal lymph nodes were harvested under sterile conditions, and single cell suspensions were prepared. Flow cytometry was performed on a BD LSRFortessa and analyzed using BD FACSDiva 8.0.2 software.
The following primary antibodies were used for staining: aqua fluorescent reactive dye (Invitrogen, Cat# L34966 A), FITC anti-mouse CD3 (Biolegend, Cat# 100203, clone: 17A2), AF700 anti-mouse CD8a (Biolegend, Cat# 100730, clone: 53–6.7), FITC anti-mouse CD11c (Biolegend, Cat# 117306, clone: N418), PerCP anti-mouse CD45 (BD Pharmingen, Cat# 557235, clone: 30-F11), PE anti-mouse CD80 (Biolegend, Cat# 104707, clone: 16–10A1), APC anti-mouse CD86 (Biolegend, Cat# 105012, clone: GL-1), and AF594 anti-mouse/human Ki67 (Biolegend, Cat# 151214, clone: 11F6)

Wilhelm J., Quiñones-Pérez M., Wang J., Wang X., Basava V.S, & Gao J. (2020). Antigen Folding Improves Loading Efficiency and Antitumor Efficacy of PC7A Nanoparticle Vaccine. Journal of controlled release : official journal of the Controlled Release Society, 329, 353-360.

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