Zombie aqua staining

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Zombie Aqua staining is a fluorescent stain used to detect cell death in flow cytometry applications. It binds to dead cells and emits a fluorescent signal that can be measured using a flow cytometer.

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Zombie Aqua

9 protocols using zombie aqua staining

Isolation and Characterization of Cardiac and Splenic Immune Cells

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Heart cell suspensions and splenocytes were prepared as previously described [12 , 13 ]. Cardiac macrophages were gated as CD45⁺Aqua⁻Ly6G⁻CD64⁺, splenic neutrophils were gated as CD45⁺Aqua⁻Ly6G⁺ and splenic B cells were gated as CD45⁺Aqua⁻CD19⁺. Antibody dilutions and specifications are described in “Flow cytometry” section. Cell viability was assessed by Zombie Aqua staining (BioLegend; Cat#423102; dilution 1:100). Cells were isolated through FACS sorting on a BD FACS Aria-III (nozzle 100μm).
For culture of primary macrophages in B cell conditioned media experiment, discrimination of live vs dead cells was achieved using Vibrant DyeCycle Violet Ready Flow dye (ThermoFisher) to mark viable cells and Propidium Iodide to stain dead cells. CD45⁺Aqua⁻Ly6G⁻CD64⁺ cells and CD45⁺CD19⁺ cells were isolated with a MoFlow sorter.

Rocha-Resende C., Pani F, & Adamo L. (2021). B cells modulate the expression of MHC-II on cardiac CCR2− macrophages. Journal of molecular and cellular cardiology, 157, 98-103.

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Comprehensive Immune Cell Profiling

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Splenocytes were stimulated using Cell Activation Cocktail (with Brefeldin A) (BioLegend) at 37 °C and 5% CO₂ for 5 h. Cells were then collected, and dead cells were stained with Zombie Aqua staining (BioLegend). The Intracellular Fixation & Permeabilization Buffer Set (eBioscience) was used for intracellular cytokine staining. Cells were fixed for 20 min at 37 °C. Permeabilized cells were then stained with IL-6-APC (clone RM4-4, dilution 1:200, BioLegend, Cat 116008). For Foxp3 staining to evaluate regulatory T cells, True-Nuclear Transcription Factor Buffer Set (BioLegend) was used. Briefly, splenocytes or draining lymph node lymphocytes were collected and fixed for 45 min, washed with permeabilization buffer, and stained with Alexa Fluor 488-conjugated anti-Foxp3 and isotype control antibodies (eBioscience), followed by surface staining with CD4-Pacific blue (clone RM4-4, dilution 1:200, Cat 116008), CD25-PE (clone 7D4, dilution 1:200, BD biosciences, Cat 558642), CD3ε -PE/Dazzle 594 (clone 145-2C11, dilution 1:100, Cat 100347) and CD45-APC (clone 30-F11, dilution 1:400, BioLegend, Cat 103112) at 4 °C for 30 min. Data were acquired using FACS LSR Fortessa (Becton, Dickinson and Inc.) and analyzed using the FlowJo Software 10.6.2 (BD Biosciences).

Chen W., Toda E., Takeuchi K., Sawa Y., Wakamatsu K., Kuwahara N., Ishikawa A., Igarashi Y., Terasaki M., Kunugi S., Terasaki Y., Yamada K., Terashima Y, & Shimizu A. (2024). Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism. Communications Biology, 7, 488.

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Flow Cytometry-based Cell Death and Cycle Analysis

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For cell death analysis, cells were stained with Pacific blue-labeled annexin V from Biolegend (San Diego, CA, USA) and propidium iodide (PI) (Sigma-Aldrich) for 15 min in the dark and analyzed by flow cytometry (BD FACSVerse, BD Biosciences). For cell cycle analysis, the cells were fixed with ice-cold 70% ethanol, stained with a solution containing PI, RNase A and Triton X-100 (all Sigma-Aldrich) in PBS for 30 min at 4 °C and then analyzed by flow cytometry (BD FACSVerse analyzer, BD, Allschwil, Switzerland).
For TIL analysis, samples were preincubated with anti-mouse CD16/CD32 (BioLegend) to block Fc receptors and dead cells were excluded by Zombie Aqua staining (Biolegend). The following antibodies were used: anti–CD45-Pblue, anti–CD4-AF700, anti–NKp46-APC, anti–CD11b-PE and anti-F4/80-APC-Cy7 from BioLegend (San Diego, CA) or anti–CD8-PE (BD Pharmingen), and isotype-matched antibodies from Sigma-Aldrich. Data acquisition was done on a BD FACSVerse and data analysis with FlowJo (Tree Star, Stanford, CA, USA).

Silginer M., Papa E., Szabó E., Vasella F., Pruschy M., Stroh C., Roth P., Weiss T, & Weller M. (2023). Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition. Acta Neuropathologica Communications, 11, 41.

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Immune Cell Profiling by Flow Cytometry

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Cells were pre-incubated with anti-CD16/32 antibody (Biolegend) for 5 min at 4 °C. Fluorochrome-labeled antibodies were added and incubated for 30 min at 4 °C. The following antibodies were used for stainings: anti-CD45 (30-F11), anti-CD19 (1D3) purchased from BD Bioscience, anti-CD4 (GK1.5), anti-CD8a (53–6.7), anti-TCRb (H57-597), anti-CD11b (M1/70), anti-F4/80 (BM8), anti-NK1.1 (PK136), anti-Ly6C (HK1.4) and anti-Ly6G (1A8) all purchased from Biolegend. Zombie Aqua staining (Biolegend) was used to exclude dead cells during flow cytometry. Data was acquired on a BD LSR Fortessa Cytometer (BD Bioscience).

Schömig T., Diefenhardt P., Plagmann I., Trinsch B., Merz T., Crispatzu G., Unnersjö-Jess D., Nies J., Pütz D., Sierra Gonzalez C., Schermer B., Benzing T., Brinkkoetter P.T, & Brähler S. (2024). The podocytes’ inflammatory responses in experimental GN are independent of canonical MYD88-dependent toll-like receptor signaling. Scientific Reports, 14, 2292.

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Cell Viability Assessment via Zombie Aqua Staining

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Cell viability was determined by Zombie Aqua staining (29 (link)) (Biolegend, San Diego, CA), then incubated with fluorophore-conjugated antibodies at 1:10 dilution for 15 minutes at 4 C, then washed twice and resuspended in 2% PFA solution until analysis. Intracellular flow cytometry was performed (30 (link)) and analyzed using an LSR Fortessa cytometer (BD Biosciences), and FlowJo version 10 software (Ashland, OR). Median fluorescence intensity (MFI) fold change calculated by normalizing after subtracting the isotype control MFI.

Concha-Benavente F., Srivastava R.M., Trivedi S., Lei Y., Chandran U., Seethala R.R., Freeman G.J, & Ferris R.L. (2015). Identification of the cell-intrinsic and extrinsic pathways downstream of EGFR and IFNγ that induce PD-L1 expression in head and neck cancer. Cancer research, 76(5), 1031-1043.

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Monocyte-Mediated Tumor Cell Proliferation

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Monocytes were enriched from bone marrow cells by MACS negative selection for CD3, B220, Ly-6G, NK1.1, Ter119, CD49b, and c-kit. The percentage of Ly-6C^hi Ly-6G⁻ monocytes were >80%. Monocytes (1 × 10⁵) and 100 DsRed⁺ LLC tumor cells were co-cultured for 24 h. Proliferating tumor cell clusters containing more than 10 cells were enumerated by fluorescent microscopy. For flow cytometry analysis, cultured cells were harvested with trypsin-EDTA after 3 days of culture. Cells were stained with CD45-FITC (clone 102, BioLegend), then stained with annexin V-APC in binding buffer (BD biosciences). For detection of DNA synthesis, 5-ethynyl-2´-deoxyuridine (EdU) was added to the culture in the final hour of incubation. Intracellular EdU was stained using Click-iT^® EdU Alexa Fluor™ 647 flow cytometry assay kits (Thermo Fisher Scientific) according to manufacturer’s instructions. Flow cytometry data were obtained by gating CD45(−) DsRed(+) cells and analyzing annexin V-positive cells or EdU-positive cells. Dead cells were excluded by Zombie Aqua staining (BioLegend). Stained cells were measured using Flow-Count™ Fluorospheres (Beckman Coulter) for the determination of absolute counts.

Terashima Y., Toda E., Itakura M., Otsuji M., Yoshinaga S., Okumura K., Shand F.H., Komohara Y., Takeda M., Kokubo K., Chen M.C., Yokoi S., Rokutan H., Kofuku Y., Ohnishi K., Ohira M., Iizasa T., Nakano H., Okabe T., Kojima H., Shimizu A., Kanegasaki S., Zhang M.R., Shimada I., Nagase H., Terasawa H, & Matsushima K. (2020). Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties. Nature Communications, 11, 609.

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Multiparametric Flow Cytometry Analysis

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After saturation with anti-CD32/CD16, cells from BAL fluids were incubated with monoclonal antibodies reactive to CD3 (17A2, PerCP Cy5.5, BioLegend), CD4 (GK1.5, FITC, BD Biosciences), CD8 (Ly2, 53–6.7, APC, BioLegend) and CD335 (NKp46) (29A1.4, BV605, SONY) or CD49b (DX5, biotin, BD Biosciences). PE-conjugated streptavidin (BD biosciences) was used to label biotin CD49b. Dead cells were eliminated by a Zombie Aqua staining (BioLegend). Data were acquired with a FACScalibur or a FACS-Fortessa (BD biosciences) and analyzed with the FlowJo Sofware v7.5 (Tree Star Inc.).

Vidy A., Maisonnasse P., Da Costa B., Delmas B., Chevalier C, & Le Goffic R. (2016). The Influenza Virus Protein PB1-F2 Increases Viral Pathogenesis through Neutrophil Recruitment and NK Cells Inhibition. PLoS ONE, 11(10), e0165361.

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Multiparametric Analysis of Tumor-Infiltrating Lymphocytes

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Single cell suspensions from peripheral blood and tumor tissue were surface labeled using Abs mentioned above for 15 mins at room temperature (RT). For intracellular cytokine production of IFN- γ, IL-10 and Granzyme B, TILs were stimulated in vitro with anti-CD3/CD28 beads and protein transport inhibitor (BD GolgiPlug™) for 6h. Intracellular staining for Foxp3, IL-10, granzyme B, ki67 and IFN- γ was performed as follows. After PBMC or TIL were stained with mAb for surface markers, they were fixed and permeabilized 30 mins (eBioscience, San Diego, USA). After washing, cells were subjected to intracellular staining for 30 mins at RT. Cell viability was determined by Zombie Aqua staining (Biolegend, San Diego, USA) or Fixable Viability Dye eFluor 780 (eBioscience, San Diego, USA). Samples were analyzed using an LSR Fortessa cytometer (BD Biosciences), and FlowJo version 10 software.

Liu Z., McMichael E.L., Shayan G., Li J., Chen K., Srivastava R., Kane L.P., Lu B, & Ferris R.L. (2018). Novel effector phenotype of Tim-3+ regulatory T cells leads to enhanced suppressive function in head and neck cancer patients. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(18), 4529-4538.

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Quantification of ORFV-infected HeLa cell immunogenicity

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Cell viability of HeLa-K^b cells 20 h post infection was determined by Zombie Aqua staining (BioLegend, San Diego, CA, USA) followed by flow cytometry. The infection rate was calculated by flow cytometric detection of mCherry or Green Fluorescent Protein (GFP) expressing cells. Quantification of H-2K^b molecules on the surface of ORFV-infected HeLa-K^b cells was done using QIFIKIT (Dako, Glostrup, Denmark) according to the manufacturer’s protocol. Detection of Ova_257-264 SIINFEKL peptide on the surface of ORFV-infected cells was performed after staining with Allophycocyanin (APC) anti-mouse H-2K^b bound to SIINFEKL antibody (BioLegend, San Diego, CA, USA; clone 25-D1.16). Samples were acquired on a BD Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR, USA).

Reguzova A., Ghosh M., Müller M., Rziha H.J, & Amann R. (2020). Orf Virus-Based Vaccine Vector D1701-V Induces Strong CD8+ T Cell Response against the Transgene but Not against ORFV-Derived Epitopes. Vaccines, 8(2), 295.

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