For immune-profiling, tumors were cut into small pieces (1 mm3), treated with 1 mg/mL collagenase A (MilliporeSigma, Burlington, MA, USA) and 250 units/mL DNase I (MilliporeSigma) at 37°C for 30 min, and filtered through a 40 μm strainer. The homogenate was layered onto Lympholyte M (CEDARLANE, Burlington, NC, USA). Buffy coat was incubated with Zombie Aqua viability kit (Biolegend), Fc-block (Biolegend), followed by the primary antibodies. Following fixation with FluoroFix Buffer (Biolegend), the data were collected on the FACSCelesta instrument (BD Biosciences, San Jose, CA, USA), analyzed by the BD FACSDiva software (BD Biosciences) and the FlowJo software version 10 (BD Biosciences). To quantify mCherry expressing breast cancer cells, the lungs were digested with 62.5 μg/mL Liberase (MilliporeSigma) and 500 units/mL DNase I (MilliporeSigma). The cell suspension was incubated with Zombie Aqua viability kit (Biolegend) and analyzed using a S1000EON benchtop flow cytometer (Stratedigm, San Jose, CA, USA).
Zombie aqua viability kit
Manufactured by BioLegend
Sourced in United States
The Zombie Aqua viability kit is a fluorescence-based assay used to detect dead or dying cells. The kit utilizes a membrane-impermeable dye that specifically stains the nucleic acids of cells with compromised membranes, providing a quantitative measure of cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharide lps, by Merck Group (2 mentions)
Fc block, by BioLegend (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
β actin 13e5, by Cell Signaling Technology (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
Endotoxin free ova, by InvivoGen (1 mentions)
Stabilizing fixative, by BD (1 mentions)
Anti cd16 32, by BioLegend (1 mentions)
F ab 2 fragment goat anti mouse igm, by Jackson ImmunoResearch (1 mentions)
Sars cov 2 s protein, by GenScript (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Mhcii bv421 m5 114, by BioLegend (1 mentions)
Cd45 30 f11, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Polyclonal anti p2x7r antibody, by Abcam (1 mentions)
Oxidized atp oatp, by Merck Group (1 mentions)
Ril 13, by Thermo Fisher Scientific (1 mentions)
Fluorofix buffer, by BioLegend (1 mentions)
8 protocols using zombie aqua viability kit
1
Immune Profiling of Tumor Samples
2
Mouse Immune Cell Characterization Protocol
3
Characterization of SARS-CoV-2 Antibody Response
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N,N-(diethylamino)ethyl methacrylate (DEAEM), Pluronic F127, HEL, IgG from rat serum, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). Goat anti-mouse IgM F(ab′)2 fragment, goat anti-mouse IgM Fab fragment, and alkaline phosphatase–conjugated anti-mouse IgM were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Endotoxin-free OVA was purchased from InvivoGen (San Diego, CA). Y. pestis fusion protein F1-V (NR-4526) was obtained from the Biodefense and Emerging Infections Repository (Manassas). SARS-CoV-2 S protein was purchased from GenScript (Piscataway, NJ). Antibodies for flow cytometry [phycoerythrin (PE) anti-mouse CD19, allophycocyanin/Cyanine 7 (Cy7) anti-mouse B220, Zombie Aqua viability kit, PE-Cy7 anti-mouse Nur77, and peridinin chlorophyll protein (PerCp)–Cy5.5 anti-mouse CD3 and anti-CD16/32] and purified anti-mouse CD40 antibody were purchased from BioLegend (San Diego, CA). BD stabilizing fixative was purchased from BD Biosciences (Franklin Lakes, NJ). All other chemicals and materials were purchased from Fisher Scientific (Pittsburgh, PA).
4
Evaluating MNP Cytotoxicity on DC Subsets
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Cytotoxicity of PE-coated and uncoated MNPs on various DC subsets was evaluated using the Zombie Aqua Viability Kit (BioLegend, Fell, Germany) according to the manufacturer’s protocol. Briefly, 5 × 105 cells after MNP labeling were incubated in a 1:100 (v/v) dilution of the Zombie Aqua dye in a total volume of 50 μL PBS for 20 min at room temperature in the dark. After washing with PBS, cells were analyzed with the FACSCanto II flow cytometer, and data were evaluated using FlowJo software.
5
Detailed Immune Cell Profiling
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Cells isolated from tissues were stained with Zombie Aqua Viability Kit and for CD45 (30-F11), CD3 PE-Cy7 (17A2), CD11c PE (N418), CD11b PercP-Cy5.5 (M1/70), Ly6C A700 (HK1.4), Ly6G FITC (1A8) and MHC-II Bv421 (M5/114.15.2) (Biolegend, San Diego, CA). Another panel was used to identify activated T cells in lymph nodes and lungs: CD45 APC-Cy7 (30-F11), CD4 FITC (GK1.5), CD8 PE (YTS156.77), CD3 PercP-Cy5.5 (17A2), TNF-α APC (MP6-XT22), Ly6C A700 (HK1.4) and IFN-γ Bv421 (XMG1.2). All antibodies were used in a 1:200 dilution, except for MHC-II that was 1:50 dilution. Antibodies were validated by Biolegend in mouse splenocytes and extensive literature has been published using these antibodies. Data was acquired on an LSR-II (BD Biosciences, San Jose, CA) using DIVA software and was analyzed with FlowJo v10.8.1. The gating strategy for myeloid cell compartment is detailed in Fig. S2d .
6
Isolation and FACS Analysis of Murine Tumor Cells
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A single cell suspension from mouse tumors was prepared and subjected to FACS analysis [52 (link)]. Briefly, mouse tumors were cut into small pieces, treated with collagenase A, and filtered through a 70 µm strainer. Leukocytes were isolated by using Fico/Lite LymphoH density gradient sedimentation (Atlanta Biologicals, Lawrenceville, GA, USA). Cells were incubated with Zombie Aqua viability kit (Biolegend, San Diego, CA, USA) to stain dead cells and Fc-block (Biolegend) to prevent nonspecific binding. Cell membrane antigens were stained with antibodies described in Supplementary Figure S7 . The cells were analyzed using a BD LSRII analyzer. Data were acquired using BD FACSCanto II (BD Biosciences, San Jose, CA, USA) and FlowJo software version 10 (Tree Star, Ashland, OR, USA).
7
Immune Cell Phenotyping Protocol
8
Immune Cell Phenotyping by Flow Cytometry
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Cells were isolated from the tissues and stained with Zombie Aqua Viability Kit and for CD45 APC-Cy7, CD3 PE-Cy7, CD64 PE-594, CD11c PE, CD11b PercP-Cy5.5, Ly6C A700, Ly6G FITC and MHC-II Bv421 (Biolegend, San Diego, CA). Other staining panels were used: CD3 PercPCy5.5, CD8 FITC, CD4 PE, CD40L PE-Cy7 and CD69 A700. Data was acquired on an LSR-II (BD Biosciences, San Jose, CA) and analyzed with FlowJo v10. Gating strategy is detailed in Supplementary Figs. S1A and S2.
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