Live dead aqua fluorescent reactive dye

Manufactured by Thermo Fisher Scientific

Sourced in United States

The LIVE/DEAD Aqua fluorescent reactive dye is a cell viability stain used to distinguish living cells from dead cells in flow cytometry applications. The dye is membrane-impermeable and can only enter cells with compromised membranes, staining dead or dying cells. This allows for the identification and quantification of live and dead cell populations within a sample.

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Live/Dead Aqua (379 citations) Live/Dead dye (97 citations) Aqua Live/Dead dye (39 citations) Fluorescent dyes (25 citations) Aqua fluorescent reactive dye (17 citations) Aqua dye (13 citations) Live/Dead fluorescent dye (5 citations) AQUA LIVE/DEAD fluorescent dye (4 citations) Live/Dead fixable aqua fluorescent reactive dye (3 citations) Aqua Fluorescent Reactive (2 citations)

7 protocols using live dead aqua fluorescent reactive dye

Isolation of Kidney Graft Immune Cells

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After sacrifice, mice were flashed with 0.9% saline through heart to remove the peripheral blood remained in the kidney allografts. Kidney graft tissues were then collected and digested using Blenzyme 4 (Roche Inc., Indianapolis, IN). Kidney suspensions were pooled and stained with Live/dead Aqua Fluorescent Reactive Dye (Life Technologies) and PE conjugated anti-mouse CD45 antibody (eBioscience). DAPI^-CD45⁺ cells were FACS-sorted on (BD Influx™ cell sorter) with a 100 µm nozzle and collected (Figure S1A).

Shen Q., Wang Y., Chen J., Ma L., Huang X., Tang S.C., Lan H., Jiang H, & Chen J. (2021). Single-Cell RNA Sequencing Reveals the Immunological Profiles of Renal Allograft Rejection in Mice. Frontiers in Immunology, 12, 693608.

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Cytotoxicity Assay for NK Cells

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Target cell killing was determined using a combination of viability stains. Cytotoxicity assays were performed using an NK cell to target cell ratio of 5:1 at 37 °C for 5 h, after which cells were stained surface markers (CD56) to discriminate NK cells from target cells, with the addition of Live/dead aqua-fluorescent reactive dye (1:200; Life Technologies) for 20 min at 4 °C (or 15 min at RT), washed in staining buffer and stained in 50 μl RPMI media plus 1 μM Yo-Pro®-3 iodide (Life Technologies) for 15′ at 37 °C. Finally, cells were washed and either directly analyzed using a BD™ LSR-II cytometer or fixed in 100 µl PFA 2–4% for 10 min at 4 °C, pelleted, washed twice with 200 µl staining buffer, and then rested at 4 °C until analyzed at the LSR-II machine.

Goodridge J.P., Jacobs B., Saetersmoen M.L., Clement D., Hammer Q., Clancy T., Skarpen E., Brech A., Landskron J., Grimm C., Pfefferle A., Meza-Zepeda L., Lorenz S., Wiiger M.T., Louch W.E., Ask E.H., Liu L.L., Oei V.Y., Kjällquist U., Linnarsson S., Patel S., Taskén K., Stenmark H, & Malmberg K.J. (2019). Remodeling of secretory lysosomes during education tunes functional potential in NK cells. Nature Communications, 10, 514.

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Quantification of Tetramer-Specific CD8+ T Cells

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PBMC and tissue-derived MNC were thawed and subjected to Live/Dead Aqua fluorescent reactive dye (ThermoFisher) in PBS for 30 mint at RT. The cells were washed cells with RPMI + 10% FBS and subsequently incubated with 3µg/mL A01-CM9 tetramers (NIH Tetramer Core Facility and MBL International) in PBS + 2% FBS at room temperature for 35 min at 2 × 10⁶ cells/100 µL in polypropylene FACS tubes. The cells were then incubated at 4 ^°C with antibodies directed to cell surface markers for additional 30 min. After 2 washes with PBS + 2% FBS, the cells were fixed with 200 µL of stabilizing fixative (BD Biosciences) at room temperature. Tetramer + CD8 + T cells were quantified with the aid of a Fortessa flow cytometer (BD) by gating on live, singlets, CD45 + , CD8 + CD3 + CD14- CD20- CD4- cells present in the lymphocyte gate (CD45-PerCP, BD Biosciences, catalog number 558411, 1:10 dilution; CD8-BUV395, BD Biosciences, catalog number 563795, 1:50 dilution; CD3-FITC BD Biosciences, catalog number 556611, 1:5 dilution; CD14-PE-Cy7, BD Biosciences, catalog number 557742, 1:100 dilution; CD20-PE-Cy7, BD Biosciences, catalog number 560735, 1:50 dilution; CD4-BV785, Biolegend, catalog number 317442, 1:100 dilution).

Colonna L., Peterson C.W., Schell J.B., Carlson J.M., Tkachev V., Brown M., Yu A., Reddy S., Obenza W.M., Nelson V., Polacino P.S., Mack H., Hu S.L., Zeleski K., Hoffman M., Olvera J., Furlan S.N., Zheng H., Taraseviciute A., Hunt D.J., Betz K., Lane J.F., Vogel K., Hotchkiss C.E., Moats C., Baldessari A., Murnane R.D., English C., Astley C.A., Wangari S., Agricola B., Ahrens J., Iwayama N., May A., Stensland L., Huang M.L., Jerome K.R., Kiem H.P, & Kean L.S. (2018). Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation. Nature Communications, 9, 4438.

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Cryopreserved PBMC Immunophenotyping

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Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient (Axis-Shield, Oslo, Norway) and cryopreserved in freezing medium (10% DMSO, 90% fetal calf serum; Lonza, CA, USA) in liquid nitrogen until used. PBMCs were thawed, washed twice with phosphate-buffered saline (PBS, Lonza, CA, USA), and stained at room temperature for 15 min with the following antibodies: BV570-CD3 (clone UCHT1), APC-Cy7-CD4 (clone A161A1), and Alexa Fluor 700-CD8 (clone RPA-T8) (BD Biosciences, San Jose, CA, USA). Viability was determined using live/dead aqua fluorescent reactive dye (Thermo Fisher Scientific, CA, USA). Cells were washed and fixed in 300 μl of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and acquired immediately in a FACS Symphony cytometer (BD, Biosciences). Fluorescence minus one stained tubes were used as gating controls. Data were analyzed using FlowJo v.10 software (FlowJo LLC, Ashland, OR, USA) (Supplementary Figure S1).

Guzmán-Guzmán I.P., Nogueda-Torres B., Zaragoza-García O., Navarro-Zarza J.E., Briceño O., Pérez-Rubio G., Falfán-Valencia R., Gutiérrez-Pérez I.A, & Parra-Rojas I. (2022). The Infection, Coinfection, and Abundance of Intestinal Protozoa Increase the Serum Levels of IFABP2 and TNF-α in Patients With Rheumatoid Arthritis. Frontiers in Medicine, 9, 846934.

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Isolation and Characterization of CD8+ T Cell Subsets

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For the eight elderly donors, PBMCs were isolated from fresh blood samples using density gradient centrifugation with Ficoll (GE Healthcare). CD3⁺ T cells were enriched from PBMCs by immunomagnetic selection using CD3 MicroBeads (Miltenyi Biotec, Auburn, CA). Cells were stained in the dark for 15 min with the following anti-human Abs: CD45RO PE-Cy7 (BD Biosciences, San Jose, CA), CD3 Alexa Fluor 700 (BD Biosciences), CD62L-PE (BD Biosciences), CD45RA-allophycocyanin (BD Biosciences), CD8-Pacific Blue (BD Biosciences), CD4 allophycocyanin-Cy7 (BD Biosciences), and LIVE/DEAD Aqua fluorescent reactive dye (Invitrogen, Grand Island, NY).
CD8⁺ T cell subsets were isolated using the BD FACSAria cell-sorting system (BD Biosciences), including CD8⁺CD45RA⁻CD45RO⁺ (for CD8⁺ memory), CD8⁺CD45RA⁺CD45RO⁻CD62L^hi (CD8⁺ naive), and CD8⁺CD45RA⁺CD62L^lo/− (CD8⁺ T_EMRA). FlowJo (TreeStar, Ashland, OR) analysis was used to determine the proportions of the different subsets as a fraction of total CD8⁺ T cells.

Lindau P., Mukherjee R., Gutschow M.V., Vignali M., Warren E.H., Riddell S.R., Makar K.W., Turtle C.J, & Robins H.S. (2018). Cytomegalovirus Exposure in the Elderly Does Not Reduce CD8 T Cell Repertoire Diversity. The Journal of Immunology Author Choice, 202(2), 476-483.

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Multiparametric Flow Cytometry Analysis of B Cell Subsets

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Previously frozen PBMCs were incubated in 1 mL of pre-warmed complete media (RPMI 1640 supplemented with 10% FBS and 2mM L-glutamine) containing 20 nM of MitoTracker Green (Invitrogen) at 37°C for 30 mins. Cells were collected by centrifugation then resuspended in 10 μL of warm complete medium and incubated at 37°C for 30 mins. Next, the cells were stained on ice for 30 mins in 100 μLs FACS buffer (PBS + 0.5% BSA) containing 5% normal mouse serum, 5% normal rat serum and the following fluorochrome-conjugated mouse anti-human monoclonal antibodies: PE-IgD (IA6-2, BD Biosciences), PE-A610-CD24 (SN3, Invitrogen), PE-Cy5-CD21 (B-ly4, BD), PerCP-Cy5.5-CD38 (HIT2, BD), PE-Cy7-CD23 (EBVCS2, eBioscience), Pacific Blue-CD3 (SP34-2, BD), Qdot605-CD27 (CLB-27/1, Invitrogen), allophycocyanin-CD95 (DX2, BD), allophycocyanin-Cy7-CD19 (SJ25C1, BD Biosciences), as well as the biotinylated 9G4 rat anti-human Ig idiotype antibody, which was detected by a further staining with 100 μL of streptavidin-Alexa680 (Invitrogen) at a 1:500 dilution in FACS buffer on ice for 30 mins. Cells were washed with 3 mLs PBS and stained with 1 mL LIVE/DEAD aqua-fluorescent reactive dye (Invitrogen) at a 1:1000 dilution in PBS on ice for 30 mins. After resuspension in FACS buffer, cells were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Newell K.A., Asare A., Sanz I., Wei C., Rosenberg A., Gao Z., Kanaparthi S., Asare S., Lim N., Stahly M., Howell M., Knechtle S., Kirk A., Marks W.H., Kawai T., Spitzer T., Tolkoff-Rubin N., Sykes M., Sachs D.H., Cosimi A.B., Burlingham W.J., Phippard D, & Turka L.A. (2015). Longitudinal Studies of a B Cell Derived Signature of Tolerance in Renal Transplant Recipients. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(11), 2908-2920.

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Evaluating Cytotoxicity of DV2 Peptide

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The cytotoxic effects of the DV2 peptide were evaluated by measuring cell metabolic activity as well as cell viability. Cell proliferation was assessed using the reagent WST-1 (Roche) according to the manufacturer’s instructions, and the number of live cells was determined by flow cytometry. Briefly, Vero CCL-81 cell monolayers established in 96-well plates were incubated at 37 °C for 24 h with increasing concentrations of the peptide ranging from 0.75 to 24 μM. Equivalent volumes of DMSO were used as the control. The WST-1 reagent was added to the wells, which were then incubated for 1 h at 37 °C. The absorbance was measured using a microplate reader at 440 nm. For flow cytometry analysis, live/dead aqua fluorescent reactive dye (Invitrogen) was added to the wells after a washing step for incubation for 30 min. at room temperature. The cells were then acquired on a LSR FortessaTM cytometer (BD, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (version 10, Tree Star, San Carlo, CA, USA).

de Castro-Amarante M.F., Pereira S.S., Pereira L.R., Santos L.S., Venceslau-Carvalho A.A., Martins E.G., Balan A, & de Souza Ferreira L.C. (2023). The Anti-Dengue Virus Peptide DV2 Inhibits Zika Virus Both In Vitro and In Vivo. Viruses, 15(4), 839.

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