Zombie near ir

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Zombie Near-IR is a fluorescent dye that can be used to detect cell death in various experimental systems. The dye binds to exposed DNA in dying or dead cells, emitting a near-infrared signal that can be detected using appropriate instrumentation.

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7 protocols using zombie near ir

Multi-Marker Immunophenotyping of Lung Cells

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Single cell suspensions were stained with Zombie Near-IR (Biolegend) to distinguish live and dead cells. Cells were stained with the following antibodies (Biolegend or BD Biosciences): CD3 BUV395, CD4 AF700, CD8 BV605, CD44 PE-Cy7, CD62L Pacific Blue, CD69 PE, CD103 APC, IFN-γ PE, TNF-α APC, and/or IL-2 Pacific Blue. Prior to intracellular cytokine staining, 1.5E6 lung cells were stimulated for 5 hours with Influenza A NP or SCV2 M, N, or S+ Peptivator pools (Miltenyi Biotec) or for 4 hours with PMA/ionomycin (Biolegend). During the last 4 hours of culture, brefeldin A (Biolegend) was added to inhibit cytokine secretion. Data acquisition was performed on a Symphony Flow Cytometer (BD Biosciences) and data analyzed using FlowJo 10 (BD Biosciences). The gating scheme utilized for analysis, including assessment of cytokine production among cells stimulated with peptide pools is shown in Supplemental Figs. 1 and 2.

Roberts L.M., Jessop F., Wehrly T.D, & Bosio C.M. (2021). Lung-resident T cells elicited by SARS-CoV-2 do not mediate protection against secondary infection. Journal of immunology (Baltimore, Md. : 1950), 207(10), 2399-2404.

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Functional Profiling of Antigen-Specific CD8+ T Cells

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Dextramer‐selected P1‐specific CD8⁺ T cells were co‐cultured with the HLA‐A*02 AML cell line THP1 (DSMZ, GE, EU) in a ratio 10:1. T2 cells loaded with P1 or the irrelevant HERV‐derived peptide (SMDDQLNQL) were used as positive and negative controls of specificity, respectively. After 1 h of co‐culture, Golgi Plug (BD, NJ, US) and CD107a antibody (BD, NJ, US) were added into wells and co‐cultures were maintained for further 4 h. An anti‐human MHC I blocking Ab (Clone W6/32, InVivoMab, BioXcell, NH, US) was added in selected wells for 1 h before co‐culture (50 μg/ml) and maintained at 10 μg/ml during the entire time of the co‐culture to neutralize the HLA‐A*02 dependent T cell activation, as control. Viability (Zombie NearIR, Biolegend, CA, US), extracellular (CD3, BV421/ CD8, FITC both Biolegend, CA, US), intracellular staining (IFN‐γ PE, Biolegend, CA, US and TNF‐α Pe‐Cy7, BD, NJ, US) and fixation were then performed and samples were analyzed on a LSR Fortessa (BD, NJ, US).

Alcazer V., Bonaventura P., Tonon L., Michel E., Mutez V., Fabres C., Chuvin N., Boulos R., Estornes Y., Maguer‐Satta V., Geistlich K., Viari A., Metzeler K.H., Hiddemann W., Batch A.M., Herold T., Caux C, & Depil S. (2022). HERVs characterize normal and leukemia stem cells and represent a source of shared epitopes for cancer immunotherapy. American Journal of Hematology, 97(9), 1200-1214.

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Multiparameter Flow Cytometry Analysis

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Single cell suspensions were stained with Zombie Near-IR (Biolegend) to distinguish live and dead cells according to the manufacturer’s instructions. Cells were stained with the following antibodies: CD3 BUV395, CD4 AF700, CD8 BV605, CD44 PE-Cy7, CD45.1 PE, and CD62L Pacific Blue in the presence of 2.4G2 hybridoma supernatant (cell line kindly provided by Dr. Jeffrey Frelinger). All antibodies were purchased from BD Biosciences or Biolegend and titrated on normal splenocytes prior to use. Data acquisition was performed on a Symphony Flow Cytometer (BD Biosciences) and data analysis was completed using FlowJo 10 (BD Biosciences). The gating scheme utilized for analysis is shown in Supplemental Figure 1.

Roberts L.M., Wehrly T.D., Leighton I., Hanley P., Lovaglio J., Smith B.J, & Bosio C.M. (2022). Circulating T cells are not sufficient for protective immunity against virulent Francisella tularensis. Journal of immunology (Baltimore, Md. : 1950), 208(5), 1180-1188.

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Comprehensive Immune Profiling of Btnl1-/- Mice

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Comprehensive immunophenotyping of Btnl1-/- mice was performed using a platform developed by the Wellcome Trust Infection and Immunity Immunophenotyping (3i) consortium (www.immunophenotyping.org). In brief, Spleen and MLN were digested with collagenase (1mg/ml)/DNAse (0.1 mg/ml) in 2% FCS PBS (+ Ca/Mg) for 20 minutes at 37°C and filtered through 30μm cell strainers. Cells were plated on 96 well V-bottom plates, washed in PBS and stained with Zombie Near-IR (Biolegend) for live/dead discrimination. Antibody stains were performed at 4°C for 20mins. Full details regarding phenotyping panels are included in Table S3. Samples were acquired on a BD LSR Fortessa X-20 equipped with 405nm (40mW), 488nm (50mW), 561nm (50mW), and 640nm (100mW) lasers.

Di Marco Barros R., Roberts N.A., Dart R.J., Vantourout P., Jandke A., Nussbaumer O., Deban L., Cipolat S., Hart R., Iannitto M.L., Laing A., Spencer-Dene B., East P., Gibbons D., Irving P.M., Pereira P., Steinhoff U, & Hayday A. (2016). Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific γδ T Cell Compartments. Cell, 167(1), 203-218.e17.

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Lung Immune Cell Profiling by Flow Cytometry

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Lungs were aseptically removed and placed in ice-cold PBS before processing for flow cytometry. Lungs were then minced and digested using Liberase TM (Sigma-Aldrich) as described previously (54 (link)). ACK lysis buffer was used to lyse the red blood cells, and then total number of viable cells enumerated using trypan blue exclusion. Cell counts were obtained using a TC20 automated cell counter (Bio-Rad). Single-cell suspensions were then stained with Zombie Near-IR (BioLegend) to determine live and dead cells and with the following antibodies for characterization of innate immune populations: CD45 BUV385, CD11b BV510, CD11c–PerCP-Cy5.5, CX3CR1 BV785, SiglecF-PE, CD103 BV711, Ly6C-APC, Ly6G AF700 (BioLegend). Data acquisition was conducted on a Symphony Flow Cytometer (BD Biosciences) and data analyzed using FlowJo 10 (BD Biosciences). The gating scheme used for determining lung cell populations is provided in Supplemental Figure 3.

Jessop F., Schwarz B., Scott D., Roberts L.M., Bohrnsen E., Hoidal J.R, & Bosio C.M. (2022). Impairing RAGE signaling promotes survival and limits disease pathogenesis following SARS-CoV-2 infection in mice. JCI Insight, 7(2), e155896.

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Multi-parameter Flow Cytometry Profiling

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Single cell suspensions generated as described above were phenotyped for specific cellular populations using flow cytometry. To identify live versus dead cells, single cell suspensions were stained with Zombie-Near-IR (Biolegend) according to manufacturer’s instructions. Lung cells were then stained with the following antibodies CD11b BUV510, CD11c PerCP-Cy5.5, CD45 BUV395, CD103 BV711, CD115 BV065, CD206 PECy7, CX3CR1 BV785, Ly6C APC, Ly6G AF700, F4/80 PacBlue, and SiglecF PE in the presence of 2.4G2 hybridoma supernatant to block non-specific staining (anti-CD16/CD32; cell line provided by Dr. Jeffrey Frelinger, University of Arizona). Adipose cells were stained with the following antibodies: CD11b BV510, CD45 BUV395, Ly6C PE/Dazzle594, F4/80 AF700, CD64 PerCpCy5.5, CD9 PeCy7 in the presence of 2.4G2 hybridoma supernatant. All antibodies were purchased from BD Biosciences or Biolegend. Cells were stained for 20 minutes on ice and then washed twice to remove unbound antibody. Samples were then fixed with 3% paraformaldehyde, washed, and resuspended in PBS/2%FBS prior to analysis. Acquisition of cellular populations was performed on a Symphony Flow Cytometer (BD Biosciences) and data was analyzed using FlowJo 10 software (BD Biosciences). Representative gating schemes for the lung and adipose tissue are depicted in Supplemental Figure 1.

Schwarz B., Roberts L.M., Bohrnsen E., Jessop F., Wehrly T.D., Shaia C, & Bosio C.M. (2022). Contribution of lipid mediators in divergent outcomes following acute bacterial and viral lung infections in the obese host. Journal of immunology (Baltimore, Md. : 1950), 209(7), 1323-1334.

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Comprehensive Immunophenotyping of Btn6-/- Mice

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Comprehensive immunophenotyping of Btn6^−/− mice was performed using a platform developed by the Wellcome Trust Infection and ImmunityImmunophenotyping (3i) consortium (www.immunophenotyping.org)^{53 (link)}. In brief, Spleen and MLN were digested with collagenase (1 mg/ml)/DNAse (0.1 mg/ml) in 2% FCS PBS (+Ca/Mg) for 20 min at 37 °C and filtered through 30 μm cell strainers. Cells were plated on 96-well V-bottom plates, washed in PBS and stained with Zombie Near-IR (Biolegend) for live/dead discrimination. Antibody stains were performed at 4 °C for 20 min. Full details regarding phenotyping panels are included in Table S1. Samples were acquired on a BD LSR Fortessa X-20 equipped with 405 nm (40 mW), 488 nm (50 mW), 561 nm (50 mW) and 640 nm (100 mW) lasers.

Jandke A., Melandri D., Monin L., Ushakov D.S., Laing A.G., Vantourout P., East P., Nitta T., Narita T., Takayanagi H., Feederle R, & Hayday A. (2020). Butyrophilin-like proteins display combinatorial diversity in selecting and maintaining signature intraepithelial γδ T cell compartments. Nature Communications, 11, 3769.

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