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Zombie aqua viability stain

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Zombie Aqua is a fluorescent dye used to detect cell viability. It selectively stains dead cells, allowing for the identification of viable and non-viable cell populations.

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Lab products found in correlation

Cytoflex s cytometer, by Beckman Coulter (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Cd56 pe cy7, by BD (2 mentions) Alexafluor 700 cd3, by BioLegend (1 mentions) Cd19 apc cy7, by BioLegend (1 mentions) Ab46074, by Abcam (1 mentions) Dy279, by R&D Systems (1 mentions) Fc block, by BD (1 mentions) Legendplex cytokine bead array, by BioLegend (1 mentions) Cd45 apc, by BioLegend (1 mentions) Fc block, by BioLegend (1 mentions) Ls column, by Miltenyi Biotec (1 mentions) Goat anti mouse igg1 pe cy7 secondary, by BioLegend (1 mentions)

Close spelling variants of this product

Zombie Aqua Fixable Viability stain Zombie aqua cell viability stain Zombie Aqua stain Zombie Aqua

7 protocols using zombie aqua viability stain

1

Immune Cell Phenotyping Protocol

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Staining was performed using antibodies listed in Table S2 at a 1:200 dilution as well as the Zombie Aqua viability stain (BioLegend). All intracellular staining was performed using the Foxp3 Transcription Factor Staining Buffer Set (eBioscience). Samples were acquired on CytoFlex S cytometer (Beckman Coulter) and data was analyzed using FlowJo software (BD Biosciences).
Mowat C., Dhatt J., Bhatti I., Hamie A, & Baker K. (2023). Short chain fatty acids prime colorectal cancer cells to activate antitumor immunity. Frontiers in Immunology, 14, 1190810.
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2

NK Cell Degranulation and IFN-γ Assay

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Following 6 days of culture, freshly isolated autologous NK cells were added to Mϕs at a ratio of 1:1. Cells were centrifuged at 300 g for 3 min and incubated for 16 h at 37°C with 5% CO2. Following stimulation by Mϕs, NK cells were removed by pipetting, and incubated ± K562 cells (1:1 ratio) with an antibody against the degranulation marker CD107a (BD Biosciences 328620), GolgiStop and GolgiPlug transport inhibitor for 6 h at 37°C with 5% CO2. NK cell degranulation and interferon gamma (BioLegend, 502509) production was examined by flow cytometry, identifying live NK cell populations using Zombie Aqua viability stain (BioLegend 423101), APC-CY7 CD19 (BioLegend, 302218), Alexafluor 700 CD3 (BioLegend, 300424), PE-CY7 CD56 (Becton Dickinson, 335791), and BUV395 CD16 (Becton Dickinson, 563785).
Read S.A., Wijaya R., Ramezani-Moghadam M., Tay E., Schibeci S., Liddle C., Lam V.W., Yuen L., Douglas M.W., Booth D., George J, & Ahlenstiel G. (2019). Macrophage Coordination of the Interferon Lambda Immune Response. Frontiers in Immunology, 10, 2674.
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3

Immune Cell Chemotaxis Assay

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Immune cell chemotaxis assays were performed using 1 × 106 autologous PBMCs placed in 5 μM pore size transwell inserts. Assays were performed for 24 h at 37°C and 5% CO2. Migrated cells present in the lower chamber were removed by pipetting and characterized by flow cytometry. Zombie Aqua viability stain (BioLegend 423101) and antibodies directed toward CD19 (BioLegend, 302218), CD3 (BioLegend, 300424), CD56 (Becton Dickinson, 335791), and CD14 (Becton Dickinson, 563372) were used to identify immune cell populations. All samples examined by flow cytometry have been treated with Fc block (BD, 564219) prior to staining. ELISAs for CCL2 (R&D Systems, DY279), CCL3 (R&D Systems, DY270), CCL4 (R&D Systems, DY271), CXCL10 (BioLegend, 439904), and TRAIL (Abcam, ab46074) were performed according to the manufacturers' protocols.
Read S.A., Wijaya R., Ramezani-Moghadam M., Tay E., Schibeci S., Liddle C., Lam V.W., Yuen L., Douglas M.W., Booth D., George J, & Ahlenstiel G. (2019). Macrophage Coordination of the Interferon Lambda Immune Response. Frontiers in Immunology, 10, 2674.
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4

Flow Cytometry and Cytokine Analysis

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Flow cytometry staining was performed using the antibodies in Table S2 in addition to the Zombie Aqua viability stain (BioLegend) and the Foxp3 Transcription Factor Staining Buffer Set (eBioscience). Flow cytometry was performed on a CytoFlex S cytometer (Beckman Coulter) with subsequent analysis using FlowJo (BD Biosciences).
Secretion of CCL5 and CXCL10 by dMMR and CIN CRCs into cell supernatants was analyzed using a custom LegendPlex cytokine bead array (BioLegend). Data were acquired on a CytoFlex S cytometer and analyzed in FlowJo.
Mowat C., Mosley S.R., Namdar A., Schiller D, & Baker K. (2021). Anti-tumor immunity in mismatch repair-deficient colorectal cancers requires type I IFN–driven CCL5 and CXCL10. The Journal of Experimental Medicine, 218(9), e20210108.
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5

Liver Immune Cell Isolation and Characterization

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Liver tissue was diced and incubated for 30 min at 37°C in a dissociation buffer consisting of RPMI medium containing 1 μg/ml DNAse, 0.1 mg/ml Collagenase type IV, and 100 U/ml penicillin/streptomycin. Cells were filtered through a 70 μm cell strainer and centrifuged at 50 g for 5 min to pellet hepatocytes (71 (link)). The supernatant containing liver immune cells was washed, pelleted at 400 g and frozen at −80°C until a sufficient number of samples were obtained. Fluorescence-activated cell sorting (FACS) was performed using the Becton Dickinson Influx Cell Sorter using the following panel: Zombie Aqua viability stain (BioLegend, 423102), APC CD45 (BioLegend, 304012), BUV395 CD3 (Becton Dickinson, 563546), PE-CY7 CD56 (Becton Dickinson, 335791), BV711 CD14 (Becton Dickinson, 563372), Alexafluor 488 CD68 (BioLegend, 333812), and PE IFNLR1 (BioLegend, 337804). A cell sort purity of >90% was measured during sorting.
Read S.A., Wijaya R., Ramezani-Moghadam M., Tay E., Schibeci S., Liddle C., Lam V.W., Yuen L., Douglas M.W., Booth D., George J, & Ahlenstiel G. (2019). Macrophage Coordination of the Interferon Lambda Immune Response. Frontiers in Immunology, 10, 2674.
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6

Distal Lung Cell Isolation and Profiling

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Distal lung was dissociated as above, and all incubation steps were carried out on ice. We incubated 107 cells with Fc Block (Biolegend 422301) and diluted them 1:100 in FACS buffer (2 mM EDTA and 0.2% fetal calf serum in 1× PBS, pH 7.4), for 10 min. The cells were then mixed with APC-conjugated anti-CD45 antibodies at 1 μg ml−1 in FACS buffer for 30 min, washed, and subjected to two rounds of depletion with magnetic beads according to the manufacturer’s protocol (Miltenyi: anti-human fibroblast 130–050-601, anti-CD31 130–091-935, anti-APC 130–090-855, LS column 130–042-401). Unlabelled cells were then centrifuged at 300g and labelled with a cocktail of 1 μg ml−1 of PerCP-Cy5.5 anti-EPCAM antibody and Zombie Aqua viability stain (Biolegend 423101) diluted 1:400 from stock concentration in FACS buffer.
Salahudeen A.A., Choi S.S., Rustagi A., Zhu J., van Unen V., de la O S.M., Flynn R.A., Margalef-Català M., Santos A.J., Ju J., Batish A., Usui T., Zheng G.X., Edwards C.E., Wagar L.E., Luca V., Anchang B., Nagendran M., Nguyen K., Hart D.J., Terry J.M., Belgrader P., Ziraldo S.B., Mikkelsen T.S., Harbury P.B., Glenn J.S., Garcia K.C., Davis M.M., Baric R.S., Sabatti C., Amieva M.R., Blish C.A., Desai T.J, & Kuo C.J. (2020). Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature, 588(7839), 670-675.
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7

Characterizing Pseudo-Afferent Lymph Dendritic Cells

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The cellular composition of pseudo-afferent lymph was assessed using monoclonal antibodies (CSM) expression by ALDC was then analysed using monoclonal antibodies against CD80 (IL-A159, Bio-Rad + goat anti-mouse IgG1 Pe-Cy7 secondary, Biolegend), CD86 (IL-A190, Bio-Rad, + goat antimouse IgG1 Pe-Cy7 secondary, Biolegend), CD40 (IL-A156 conjugated to FITC, Bio-Rad) and major histocompatibility complex (MHC) II (IL-A21, Bio-Rad, conjugated to AF647 in-house). All antibody conjugations were performed using Thermo Fisher Alexa Fluor Antibody Labelling Kits and following the manufacturer's instructions. Cell viability was measured using Zombie Aqua viability stain (Biolegend) according to the manufacturer's instructions. 50,000 events were collected using BD Fortessa X-20 flow cytometer and results analysed using FlowJo software (version 10.7.1). Gates were set using Fluorescence Minus One (FMO) controls (Supplementary Fig. 1).
Marzo S., Gray M., Loh W., Waddell L.A., McGuinnes C.I., Hope J.C, & Mathie H.A. (2022). Characterisation of dendritic cell frequency and phenotype in bovine afferent lymph reveals kinetic changes in costimulatory molecule expression. Veterinary immunology and immunopathology, 243.
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