Live dead aqua dead cell stain

Manufactured by Thermo Fisher Scientific

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The Live/Dead Aqua Dead Cell Stain is a fluorescent dye that enables the identification of dead cells in a sample. It binds to DNA, providing a clear distinction between live and dead cells.

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Lab products found in correlation

Cytofix cytoperm solution, by BD (3 mentions) Fitc conjugated goat anti rhesus igg h l polyclonal antiserum, by Southern Biotech (2 mentions) Anti cd27 bv421, by BD (1 mentions) Anti cd19 pe cy7, by BD (1 mentions) Mitotracker green, by Thermo Fisher Scientific (1 mentions) Mitotracker deep red, by Thermo Fisher Scientific (1 mentions) Lsrfortessa analyzer, by BD (1 mentions) Fc block, by BD (1 mentions) Anti human cd3 apc cy7, by Thermo Fisher Scientific (1 mentions) Perfix expose kit, by Beckman Coulter (1 mentions) Pe mouse igg1, by BD (1 mentions) Brefeldin a, by Merck Group (1 mentions) Anti cd28 pe, by BD (1 mentions) Anti il 17 alexa647, by BioLegend (1 mentions) Anti cd14 apc cy7, by BD (1 mentions) Anti tnf percp cy5, by BioLegend (1 mentions) Anti cd4 percp, by BD (1 mentions) Anti cd28 apc, by BD (1 mentions) Anti cd4 pe cy7, by BD (1 mentions) Anti il 17a pe, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Live/Dead Aqua (379 citations) LIVE/DEAD stain (166 citations) Aqua Live/Dead stain (60 citations) LIVE/DEAD Fixable AQUA Dead Cells Stain (2 citations)

12 protocols using live dead aqua dead cell stain

Investigating Hematopoietic Stem Cell Response to 5-FU

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NR4A1^GFP transgenic and littermate control mice received a
single intraperitoneal injection of 60 or 150mg/kg 5-FU in PBS or an equivalent
volume of sterile 1X PBS alone. Forty-two hours later, mice were euthanized by
CO₂ inhalation and bone marrow was harvested and lineage
depleted. Lin^-cells were stained for flow cytometric analysis as described
previously. LIVE/DEAD Aqua Dead cell stain (Life Technologies) was used for dead
cell exclusion.

Land R.H., Rayne A.K., Vanderbeck A.N., Barlowe T.S., Manjunath S., Gross M., Eiger S., Klein P.S., Cunningham N.R., Huang J., Emerson S.G, & Punt J.A. (2015). The orphan nuclear receptor NR4A1 specifies a distinct subpopulation of quiescent myeloid-biased long-term HSCs. Stem cells (Dayton, Ohio), 33(1), 278-288.

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Quantifying HIV-1 Variant Infectivity in JLTRG-R5 Cells

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HIV-1_NL4-3 cDNA harboring Rev-NoLS mutations was transfected into HEK293T (50% confluent) with CaCl₂ (1 μg/well) within 12-well culture plates. Viral supernatant was collected 48 h later, cleared of cell debris, and quantified for p24 production. Viral supernatant normalized to 2 ng p24 was used to infect JLTRG-R5 precultured at 1 × 10⁶ cells per well. HIV-1-exposed cells were washed thrice in 2× HBSS 24 h after infection and resupplemented with RPMI media. To assess infectivity of mutant HIV-1_NL4-3 variants, cells were collected every 5 days, stained with LIVE/DEAD aqua dead cell stain (Life Technologies), fixed in 3.7% paraformaldehyde, and measured for aqua fluorescence (405 nm excitation) with CyAn ADP 9 Color FACS analysis. Live cells were analyzed for HIV-1 infection through EGFP emission (488 nm excitation).

Arizala J.A., Takahashi M., Burnett J.C., Ouellet D.L., Li H, & Rossi J.J. (2018). Nucleolar Localization of HIV-1 Rev Is Required, Yet Insufficient for Production of Infectious Viral Particles. AIDS Research and Human Retroviruses, 34(11), 961-981.

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Flow Cytometric Analysis of PBMC Viability

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After thawing, 1 ×10⁶ PBMC were incubated with Live/Dead Aqua dead cell stain (Molecular Probes, ThermoFisher, Waltham, MA, USA) in PBS for 30 min at room temperature. Cells were incubated in FACS buffer with fluorochrome-conjugated antibodies for 30 min at room temperature; all antibodies and cytometer filters are listed in Supplementary Table 5. Data were acquired on an LSRII (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Inc; BD Biosciences, San Jose, CA, USA). Additional details provided in the Supplementary Methods. The gating schema and representative flow cytometry data are shown in Supplementary Figure 3, and marker definitions and use are shown in Supplementary Table 6.

Speake C., Bahnson H.T., Wesley J.D., Perdue N., Friedrich D., Pham M.N., Lanxon-Cookson E., Kwok W.W., Sehested Hansen B., von Herrath M, & Greenbaum C.J. (2019). Systematic Assessment of Immune Marker Variation in Type 1 Diabetes: A Prospective Longitudinal Study. Frontiers in Immunology, 10, 2023.

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Mitochondrial Dysfunction in B Cells

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Mitochondrial function was assessed using MitoTracker probes (MitoTracker Green and MitoTracker Deep Red from Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Briefly, 2 × 10⁵ harvested cells were stained with Live/Dead Aqua Dead Cell Stain (Invitrogen Molecular Probes, Eugene, OR, USA) for 20 min at room temperature (RT) followed by anti-CD19-PE-Cy7 and anti-CD27-BV421 (both from BD Biosciences, San Jose, CA, USA) staining for 15 min at RT. Then, cells were washed with PBS and stained with MitoTracker Green and MitoTracker Deep Red for the evaluation of mitochondrial mass and mitochondrial membrane potential, respectively, for 30 min at 37°C.
Reduction of mitochondrial membrane potential is a hallmark of mitochondrial dysfunction. For this reason, viable (Live/Dead⁻) naïve (CD19⁺CD27⁻) and memory (CD19⁺CD27⁺) B cells (Figure 1Ai), containing mitochondria with low membrane potential, identified as MitoTracker Deep Red^low and MitoTracker Green⁺ (Figure 1Aii), were considered B cells with dysfunctional mitochondria (CDM). Fold increase in the percentage of CDM induced by each single stimulus related to the unstimulated sample was expressed as a ratio: (single stimulus % CDM)/(unstimulated % CDM).

Berman-Riu M., Cunill V., Clemente A., López-Gómez A., Pons J, & Ferrer J.M. (2024). Dysfunctional mitochondria, disrupted levels of reactive oxygen species, and autophagy in B cells from common variable immunodeficiency patients. Frontiers in Immunology, 15, 1362995.

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Multiparametric Flow Cytometry of PBMCs

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After thawing, 1 × 10⁶ PBMC was incubated with LIVE/DEAD Aqua Dead cell stain (Molecular Probes, Eugene, Orlando, USA) for 30 min at room temperature. Fluorochrome‐conjugated antibodies were then added for 30 min at room temperature; all antibodies are listed in Supplementary table 3. Data were acquired on an LSRII (BD Biosciences, Franklin Lakes, New Jersey, USA) and analysed using FlowJo software (BD, Ashland, Oregon, USA). The gating schema and representative flow cytometry data are shown in Supplementary figure 8; the parameter name and gating details are given in Supplementary table 4.

Wesley J.D., Pfeiffer S., Schneider D., Friedrich D., Perdue N., Sehested‐Hansen B., Hagopian W, & von Herrath M.G. (2021). Peripheral autoreactive CD8 T‐cell frequencies are too variable to be a reliable predictor of disease progression of human type 1 diabetes. Clinical & Translational Immunology, 10(7), e1309.

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In Vivo Uptake of Labeled Extracellular Vesicles

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DiI‐labelled EVs or vehicle control (PBS) were injected via tail vein into male C57BL/6 mice (10 µg EV protein) 24 h prior to euthanasia. For in vivo uptake inhibition experiments, mice were subjected to HDL NP injection via tail vein (100 µM, 100 µl) 24 h prior to injection of DiI‐labelled EVs. Bone marrow cells were then isolated as described above, washed in PBS, and resuspended in FACS buffer [PBS containing 1% bovine serum albumin (BSA), 0.1% sodium azide]. Samples were stained in LIVE/DEAD Aqua Dead Cell stain (ThermoFisher) for 20 min at RT, washed in FACS buffer, and then blocked in F_c block (BD Pharmingen) for 20 min at RT. Cells were then incubated in 100 µl of fluorophore‐conjugated antibody cocktail (APC anti‐mouse/human CD11b, PE‐Cy7 anti‐mouse Ly6C, Brilliant Violet anti‐mouse Ly6G; BioLegend) (1:100 dilutions for all antibodies) for 1 h at 4°C protected from light, and then washed in FACS buffer. Samples were then analyzed using a BD LSR Fortessa Analyzer and data was processed using FlowJo software. A sample of the gating scheme is shown in Figure S1c. Experiments were performed using n = 3 mice per group.

Henrich S.E., McMahon K.M., Plebanek M.P., Calvert A.E., Feliciano T.J., Parrish S., Tavora F., Mega A., De Souza A., Carneiro B.A, & Thaxton C.S. (2020). Prostate cancer extracellular vesicles mediate intercellular communication with bone marrow cells and promote metastasis in a cholesterol‐dependent manner. Journal of Extracellular Vesicles, 10(2), e12042.

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Indirect Surface Staining of HIV-1 Env Antibodies

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Indirect surface staining was used to measure the ability of rabbit serum antibodies to bind HIV-1 Env expressed on the surface of infected cells. Binding was assessed by a previously described indirect surface staining approach (34 ) using CEM.NKR_CCR5 cells [National Institutes of Health (NIH) AIDS Reagent Program from A. Trkola (70 (link))] infected with the CH505T/F infectious molecular clone virus or mock infected cells. The cells were then incubated with a 1:100 dilution of serum samples for 2 hours at 37°C and then stained with LIVE/DEAD Aqua Dead Cell Stain (Thermo Fisher Scientific, #L34966), followed by permeabilization with Cytofix/Cytoperm solution (BD Biosciences, #554714) before staining with RD1-conjugated anti-p24 MAb KC57 (Beckman Coulter Inc., #6604667) and fluorescein isothiocyanate (FITC)–conjugated anti-rabbit IgG (H+L) (Southern Biotech, #4041-02). The result was read by flow cytometry; cells positive for serum antibody binding were defined as p24⁺ FITC⁺ and viable. Final results were reported as the change in FITC MFI or frequency of positive cells for postvaccination samples compared to the prevaccination sample after subtraction of the corresponding data from cells stained with secondary antibody alone and staining of mock-infected cells.

Chen J.L., Fries C.N., Berendam S.J., Rodgers N.S., Roe E.F., Wu Y., Li S.H., Jain R., Watts B., Eudailey J., Barfield R., Chan C., Moody M.A., Saunders K.O., Pollara J., Permar S.R., Collier J.H, & Fouda G.G. (2022). Self-assembling peptide nanofiber HIV vaccine elicits robust vaccine-induced antibody functions and modulates Fc glycosylation. Science Advances, 8(38), eabq0273.

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Plasma Binding Assay for HIV Envelope

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Indirect surface staining was used to measure the ability of plasma samples to bind HIV-1 envelope expressed on the surface of infected cells⁷². CEM.NKR_CCR5 cells were mock infected or infected with HIV-1 infectious molecular clones⁷³ expressing the 1086.C or CH505.TF envelope proteins. The cells were incubated with a 1:100 dilution of plasma samples for 2 h at 37 °C and then stained with Live/Dead Aqua Dead Cell Stain (Thermo, Fisher Scientific, Waltham, MA) to exclude dead cells from analysis. Cells were washed and then permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) prior to staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rhesus IgG(H + L) polyclonal antiserum (Southern Biotech, Birmingham, AL, Cat# 6200-02) and RD1-conjugated anti-p24 MAb KC57 (Beckman Coulter, Inc., Indianapolis, IN, Cat# 6604667). Cells positive for plasma binding were defined as viable, p24 positive, and FITC positive. Final results are reported as the FITC MFI of the live infected cell population (p24-positive cells) after subtraction of the background observed for the pre-infection (week 0) samples.

Nelson A.N., Dennis M., Mangold J.F., Li K., Saha P.T., Cronin K., Cross K.A., Kumar A., Mangan R.J., Shaw G.M., Bar K.J., Haynes B., Moody A.M., Munir Alam S., Pollara J., Hudgens M.G., Van Rompay K.K., De Paris K, & Permar S.R. (2022). Leveraging antigenic seniority for maternal vaccination to prevent mother-to-child transmission of HIV-1. NPJ Vaccines, 7, 87.

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Measuring HIV-1 Envelope Binding

Cited 2 times

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Indirect surface staining was used to measure the ability of plasma samples to bind the HIV-1 envelope expressed on the infected-cell surface by methods similar to those previously described (80 (link)). CEM.NKR_CCR5 cells were mock infected or infected with an HIV-1 infectious molecular clone (81 (link)) expressing the 1086.C or SHIV1157ipd3N4 envelope protein. The cells were incubated with a 1:100 dilution of plasma samples for 2 h at 37°C and then stained with Live/Dead Aqua Dead Cell Stain (Thermo, Fisher Scientific, Waltham, MA) to exclude dead cells from analysis. Cells were washed and then permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) prior to staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rhesus IgG (H+L) polyclonal antiserum (Southern Biotech, Birmingham, AL) and RD1-conjugated anti-p24 MAb KC57 (Beckman Coulter, Inc., Indianapolis, IN). Cells positive for plasma binding were defined as viable, p24/27 positive, and FITC positive. Final results are reported as the FITC MFI of the p24/27-positive events after subtraction of the background observed for the matched maternal prevaccination samples.

Eudailey J.A., Dennis M.L., Parker M.E., Phillips B.L., Huffman T.N., Bay C.P., Hudgens M.G., Wiseman R.W., Pollara J.J., Fouda G.G., Ferrari G., Pickup D.J., Kozlowski P.A., Van Rompay K.K., De Paris K, & Permar S.R. (2018). Maternal HIV-1 Env Vaccination for Systemic and Breast Milk Immunity To Prevent Oral SHIV Acquisition in Infant Macaques. mSphere, 3(1), e00505-17.

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T cell Activation and Signaling Assays

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CD3/CD28 activated T cells were co-cultured in 96-well plate with and without MSCs unprimed or primed in a ratio 1:1 in triplicate for 48 h. Cells were then collected, washed and phosphoflow staining was done using the Perfix Expose Kit from Beckman Coulter (B26976) per manufacturer's instructions. To be able to gate on CD4+ cells the following surface antibodies were used: Live/dead aqua dead cell stain (Thermofisher), anti-human CD3-APC/Cy7, anti-human CD4-PE. The following antibodies were used for intracellular staining: BD Phosflow™ anti-phospho-S6 ribosomal protein-V450 (S235/236), anti-phospho-MTOR pS2448-PE. The following isotype control antibodies were used: Mouse IgG1a-V450 and Mouse IgG1-PE, all purchased from BD biosciences.

Mendt M., Daher M., Basar R., Shanley M., Kumar B., Wei Inng F.L., Acharya S., Shaim H., Fowlkes N., Tran J.P., Gokdemir E., Uprety N., Nunez-Cortes A.K., Ensley E., Mai T., Kerbauy L.N., Melo-Garcia L., Lin P., Shen Y., Mohanty V., Lu J., Li S., Nandivada V., Wang J., Banerjee P., Reyes-Silva F., Liu E., Ang S., Gilbert A., Li Y., Wan X., Gu J., Zhao M., Baran N., Muniz-Feliciano L., Wilson J., Kaur I., Gagea M., Konopleva M., Marin D., Tang G., Chen K., Champlin R., Rezvani K, & Shpall E.J. (2021). Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD. Frontiers in Immunology, 12, 631353.

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