Zombie aqua

Manufactured by Thermo Fisher Scientific

Sourced in United States

Zombie Aqua is a specialized laboratory equipment designed for the analysis and processing of aqueous samples. It provides a reliable and efficient means of handling and examining water-based specimens. The core function of Zombie Aqua is to facilitate the study and characterization of water-based materials and solutions.

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Zombie Aqua Fixable Viability Kit (5 citations) Fixable Zombie Aqua (2 citations)

8 protocols using zombie aqua

Assessing Cell Viability and Apoptosis

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For viability measurement, cells were plated at 100,000 cells/mL in medium containing DMSO or 0.5 mM OSU-13 and incubated for 24, 48, and 72 hours (h) at 37°C and 5% CO₂. Afterwards, cells were stained with Zombie-aqua (Life Technologies; Carlsbad, CA, USA) according to manufacturer’s instructions. Cells were then analyzed using an Attune Nxt cytometer (Invitrogen; Waltham, MA, USA), and FlowJo software (FlowJo LLC; Ashland, OR, USA).
For apoptosis and necroptosis inhibition assays, OPM-2 cells (250,000 cells/mL) were pre-incubated for 1 h at 37^oC in medium containing 100 mM of the general caspase inhibitor Z-VAD-FMK (Sigma-Aldrich; St Louis, MO, USA) or the necroptosis inhibitor necrostatin-1s (Cell Signaling Inc.; Danvers, MA, USA). Then, cells were washed and incubated for 72 h in medium containing DMSO or 1 mM OSU-13, stained with Zombie-aqua (Life Technologies), and cell viability was assessed as previously described.

Longo L.V., Hughes T., McNeil-Laidley B., Cottini F., Hilinski G., Merritt E, & Jr. Benson D.M. (2023). TTK/MPS1 inhibitor OSU-13 targets the mitotic checkpoint and is a potential therapeutic strategy for myeloma. Haematologica, 109(2), 578-590.

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Microglia Phagocytosis Assay with Ethanol

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To evaluate changes in phagocytosis caused by ethanol challenge, microglia from young and aged mice were plated and treated as described above. Twenty-four hours after ethanol challenge, ethanol-containing media was removed from all wells and cells were then treated with FluoSpheres (580/605 fluorescent, polystyrene, 1μm in diameter, F13083, Invitrogen) at a density of 200 beads/cell resuspended in serum free media for 1 hour. FluoSphere-containing media was then removed from the wells and cells were processed for flow cytometry using markers for CD11b (1:200, 101206, Biolegend) and CD45 (1:200,103132, Biolegend) to analyze the number of CD11b⁺ CD45⁺ cells that engulfed the FluoSpheres. Cytotoxicity was determined using Zombie Aqua following treatment with ethanol or OLT1177. Twenty-four hours after ethanol challenge, cells were harvested and stained using Zombie Aqua (1:1000, Invitrogen) for 30 minutes to assess viability. They were then blocked for Fc receptors and stained for surface markers CD11b (1:200) and CD45 (1:200) for 20 minutes. Fluorescence was determined using the CytoFlex LX Flow Cytometer (Beckman Coulter) and analysis was performed using FlowJo (version 9).

Anton P.E., Nagpal P., Moreno J., Burchill M.A., Chatterjee A., Busquet N., Mesches M., Kovacs E.J, & McCullough R.L. (2024). NF-κB/NLRP3 Translational Inhibition by Nanoligomer Therapy Mitigates Ethanol and Advanced Age-Related Neuroinflammation. bioRxiv.

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Intracellular Staining and T Cell Assays

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For intracellular staining of mouse and human cells, single cell suspensions were incubated with fixable live/dead GREEN (Invitrogen) or Zombie Aqua (Invitrogen) according to the manufacturer's instructions to distinguish viable cells. Cells were washed with 2%FCS-PBS, blocked with anti-CD16/CD32 FCg II/III (WEHI antibody facility) and stained with extracellular antibodies for 30 min at 4 C. Cells were fixed and permeabilised with Foxp3/Transcription Factor Staining Kit (eBiosciences) and stained with intracellular antibodies for 30 min at 4 C. Samples were acquired on Aurora spectral unmixing cytometers (CyTEK) and analyzed using Flowjo and Cytobank.
Human lung T cell in vitro assays 96-well round bottom plates were coated with 5mg/mL anti-CD3 for a minimum of 2 h at 37 C before rinsing and removing. Human T cells were plated and stimulated with anti-CD3 (OKT3, WEHI monoclonal laboratory, 5 mg/m) and anti-CD28 (BD Biosciences, 1mg/mL) in IMDM media supplemented with 10% fetal calf serum, 1% HEPES, non-essential amino acids, glutamax, sodium pyruvate (all Gibco), 50 mM 2-mercaptoethanol (Sigma) and 100U/mL recombinant IL-2 (Peprotech). Cells were collected for flow cytometry analysis 48 and 72 h after stimulation, Golgi Stop and Golgi Plug (BD) were added to the culture media 3 h before each collection.

Weeden C.E., Gayevskiy V., Marceaux C., Batey D., Tan T., Yokote K., Ribera N.T., Clatch A., Christo S., Teh C.E., Mitchell A.J., Trussart M., Rankin L., Obers A., McDonald J.A., Sutherland K.D., Sharma V.J., Starkey G., D'Costa R., Antippa P., Leong T., Steinfort D., Irving L., Swanton C., Gordon C.L., Mackay L.K., Speed T.P., Gray D.H.D, & Asselin-Labat M.L. (2023). Early immune pressure initiated by tissue-resident memory T cells sculpts tumor evolution in non-small cell lung cancer. Cancer cell, 41(5).

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CD8+ T Cell Immunophenotyping

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After culturing cells in round-bottom 96-well plates, the CD8+ T cells were harvested, washed once with PBS, and stained with Zombie Aqua (Thermo Fisher Scientific) followed by Ab stains. Cells were fixed in 1% paraformaldehyde and analyzed using an LSRFortessa (BD Biosciences) in FACS buffer (0.1% BSA and 0.05% sodium azide in PBS). Flow cytometry samples were gated on singlets via side light scatter (SSC), and then lymphocytes were gated using forward light scatter (FSC) and SSC. Gates were drawn to exclude CD14+ CD19+ and dead cells, and expression levels and percent positive cells quantified using FlowJo v10.4 Software (BD Biosciences).

Mazahery C., Benson B.L., Cruz-Lebrón A, & Levine A.D. (2020). Chronic Methadone Use Alters the CD8+ T Cell Phenotype In Vivo and Modulates Its Responsiveness Ex Vivo to Opioid Receptor and TCR Stimuli. Journal of immunology (Baltimore, Md. : 1950), 204(5), 1188-1200.

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Apoptosis and Proliferation Analysis

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Apoptosis staining was performed using the Annexin V Apoptosis Detection Kit (eBioscience). For cell proliferation analysis, 10 7 transfected CL01 cells were washed with PBS, resuspended in 1 mL PBS containing 1 M CellTrace Violet dye (Thermo Fisher Scientific), and left in the dark at 37°C for 10 minutes. The reaction was quenched by the addition of cell culture media. The cells were washed, resuspended and cultured in a 37°C incubator until analysis via flow cytometry. Intracellular antibody staining of IgG and IgE was performed as previously described 16 using the a fixable viability stain (Zombie Aqua Thermo Fisher Scientific).

Recaldin T., Hobson P.S., Mann E.H., Ramadani F., Cousins D.J., Lavender P, & Fear D.J. (2018). miR-29b directly targets activation-induced cytidine deaminase in human B cells and can limit its inappropriate expression in naïve B cells. Molecular immunology, 101.

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CMV Peptide Stimulation of PBMCs

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De-identified peripheral blood mononuclear cells (PBMC) were collected from the Stanford Blood Center. PBMC were isolated,
frozen, and thawed as described (Higdon et al., 2016 (link)). Thawed cells were rested overnight
and stimulated with a library of 15 amino acid peptides with 11 amino acid overlap for the cytomegalovirus (CMV) gene immediate
early-1 (GenScript, Piscataway, NJ, USA) for 5 hours. Secretion of interferon (IFN)γ was measured using the Miltenyi
IFNγ Secretion Assay kit according to the manufacturer’s protocol (Miltenyi Biotech, Bergisch Gladbach, Germany).
Unstimulated cells were used as a control.
Cells were stained for 20 minutes at room temperature with fluorescently labeled antibodies for CD4 (RPA-T4), CD8 (SK1),
CD3 (OKT3), CD14 (61D3), CD16 (3G8), CD19 (HIB19) from BioLegend (San Diego, CA, USA) and Abcam (Cambridge, UK), and with Zombie
Aqua from BioLegend. Cells were resuspended in PBS with 0.5% bovine serum albumin and 2 mM EDTA. Cells were filtered through 35
μm nylon mesh into 5 mL round bottom tubes (Corning, Corning, NY, USA) immediately prior to sorting. Single color
compensation controls were prepared using UltraComp eBeads from eBioScience (Santa Clara, CA, USA) and the above antibodies, or
aliquots of cells stained with Zombie Aqua.

Higdon L.E., Cain C.J., Colden M.A, & Maltzman J.S. (2018). Optimization of single-cell plate sorting for high throughput sequencing applications. Journal of immunological methods, 466, 17-23.

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Multicolor Flow Cytometry for Dendritic Cell Subsets

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For FACS analysis as well as FACS sorting of single-cell suspensions, cells were first incubated with CD16/CD32-specific antibody (2.4G2; BD) for 10 min to block non-specific Fc-receptor interactions. A live death staining was performed using zombie aqua (eBioscience) according to the manufacturer’s instructions. To differentiate between DC subsets, cells were stained for 15 min at 4°C with combinations of antibodies specifically binding to CD11b (M1/70.15; Invitrogen), CD11c (HL3; BD), Siglec-H (eBio440c; eBioscience), CD69 (H1.2F3; BD), Clec9a (42D2; eBioscience), CD172 (SIRPα) (P84; Biolegend) and CD24 (M1/69; Biolegend). The cells were subsequently washed with 1 mL FACS buffer (PBS, 1% FCS) and then re-suspended in FACS buffer supplemented with 3% paraformaldehyde (PFA). Samples were measured using a FACS LSR II, and data were analyzed with FlowJo 7.6.5 software (TreeStar). Dead cells as well as cell duplets were excluded prior to subsequent analysis. DC subsets were sorted using the FACS Aria or FACS XDP and sorting efficiency ranged between 71.9–86.2% for the pDC, 81.4–94.4% for the CD11b-like DC and 44.54–83.2% for the CD8α-like DC.

Pfaender S., Grabski E., Detje C.N., Riebesehl N., Lienenklaus S., Steinmann E., Kalinke U, & Pietschmann T. (2016). Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner. PLoS Pathogens, 12(7), e1005736.

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Evaluating T-cell Cytotoxicity in Cancer

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Cancer cells were plated in RHS5 and, when applicable, pulsed with varying amounts of peptides. Cancer cells were then incubated with MHC-class I blocking antibody (W6/32 at 20 µg/mL), if required. T cells were cocultured with cancer cells at varying effector: target ratios. Cell Stimulation Cocktail (eBioscience) was used to stimulate the positive control cells. Cells were then stained with Zombie Aqua (eBioscience) and antibodies against CD3, CD8, IFN-g, TNFα (Biolegend) using the Intracellular Fixation and Permeabilization Buffer Set (eBioscience) following manufacturer’s instructions and analyzed by flow cytometry.
To evaluate T-cell killing capacity, cells were cocultured for 16h. T-cell killing was assessed by lactate dehydrogenase (LDH) release using the Cyto-tox nonradioactive cytotoxicity assay (Promega) as per manufacturer’s instructions, using an iMark microplate reader (Bio-Rad).

Ma R., Rei M., Woodhouse I., Ferris K., Kirschner S., Chandran A., Gileadi U., Chen J.L., Pereira Pinho M., Ariosa-Morejon Y., Kriaucionis S., Ternette N., Koohy H., Ansorge O., Ogg G., Plaha P, & Cerundolo V. (2022). Decitabine increases neoantigen and cancer testis antigen expression to enhance T-cell–mediated toxicity against glioblastoma. Neuro-Oncology, 24(12), 2093-2106.

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