Zombie red

Manufactured by BioLegend

Sourced in United States

Zombie Red is a fluorescent dye that binds to dead cells, allowing for their identification in flow cytometry analysis. It is a useful tool for assessing cell viability and apoptosis.

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Zombie Red Fixable Viability Kit (35 citations) Zombie Red fixable viability dye (16 citations) Zombie Red dye (5 citations) Zombie Red viability dye (5 citations) Zombie Red viability marker (3 citations) Zombie red staining (3 citations) Zombie RedTM Fixable Viability Kit (2 citations) Zombie NIR (Near Infra-Red) (2 citations) Zombie red fixable (2 citations) Zombie Red Dye solution (2 citations)

37 protocols using zombie red

Multi-parameter Flow Cytometry Analysis

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The following antibodies were used for flow cytometry: αCD4-APC/PE-Cy7/BV605 (GK1.5); αCD44-APC/PE-Cy7 (IM7); αCD19-AF488/APC-Cy7 (6D5); αB220-PE (RA3-6B2); αCD138-BV605 (281-2); αIgD-PE/APC-Cy7 (11-26c.2a); αIgM-e450 (II-41), αCD38-PE (90); αCD11b-PerCPCy5.5/e450(M1/70), αMHCII-BV711(M5/114.15.2), αLy6G-AF647(A18); αF4/80-Pacific blue (BM8); αCD45.1-BV785 (A20); αCD45.2-BV605 (104) (Biolegend, San Diego, CA, USA). Fc-block was used prior to surface staining. For testing viability, Propidium Iodide or Zombie Red (Biolegend) were used.
For intracellular cytokine staining, cells were first stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, Sigma-Aldrich, St. Louis, MO, USA), ionomycin (500 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) and Golgi Stop (BD Biosciences, Franklin Lakes, NJ, USA) for 3.5 hours at 37 °C. Cells were stained with fixable viability dye Zombie Red (Biolegend, San Diego, CA, USA). Cells were stained with appropriate surface antibodies listed above. Cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) before staining with αIL-17A-FITC (TC11-18H10.1), αIL10-PE/PE-Cy7 (JES5-16E3), αGM-CSF-PerCPCy5.5 (MP1-22E9) and αIFNγ-BV711 (XMG1.2) (Biolegend). All flow cytometry data were acquired with BD LSRII and analyzed with FlowJo_V10 software (Tree Star Inc., Ashland, OR, USA).

Maria Z., Turner E., Agasing A., Kumar G, & Axtell R.C. (2021). Pertussis Toxin Inhibits Encephalitogenic T-Cell Infiltration and Promotes a B-Cell-Driven Disease during Th17-EAE. International Journal of Molecular Sciences, 22(6), 2924.

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Activation-induced T Cell Immunophenotyping

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LN single cell suspensions were stained using the fixable viability dye ZombieRed (Biolegend) for 15 min. at room temperature, and added for 30 minutes at 37°C to tissue culture plates pre-coated overnight with αCD3ε (clone 145–2C11) and αCD28 (clone 37.51) antibodies (at 10 μg/ml of each antibody), or to uncoated control plates. Samples were then fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature, and permeabilized for 20 minutes through dropwise addition of 1 ml ice-cold methanol. Cells were then stained for CD90.2 (30-H12), CD4 (GK1.5), CD8α (53–6.7), CD44 (IM7) (BioLegend), Foxp3 (FJK-16s, eBioscience), pFoxo1 (Thr24)/Foxo3a (Thr32), p-c-Jun (Ser73) (D47G9) (Cell Signaling) and GFP (rabbit polyclonal Ab) (Invitrogen).

Pilato M.D., Kim E.Y., Cadilha B.L., Pruessmann J.N., Nasrallah M.N., Seruggia D., Usmani S.M., Misale S., Zappulli V., Carrizosa E., Mani V., Ligorio M., Warner R.D., Medoff B.D., Marangoni F., Villani A.C, & Mempel T.R. (2019). Targeting the CBM complex causes Treg cells to prime tumors for immune checkpoint therapy. Nature, 570(7759), 112-116.

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Isolation of Lung Epithelial Cells

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E18.5 mouse lungs were collected and a single cell suspension created as previously described (18 (link)). Cells were stained for nerve growth factor receptor (NGFR) (ab8874, 1:200; Abcam) and EpCam (47-579180, 1:200; eBioscience, San Diego, CA, USA) antibodies using Zombie Red (423109; BioLegend, San Diego, CA, USA) viability dye for dead cell exclusion on a FACSCanto cell (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometry device and using FlowJo v10.7.1 software for quantification.

Serapiglia V., Stephens C.A., Joshi R., Aydin E., Oria M., Marotta M., Peiro J.L, & Varisco B.M. (2022). Fetal Tracheal Occlusion Increases Lung Basal Cells via Increased Yap Signaling. Frontiers in Pediatrics, 9, 780166.

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Multiparameter Immune Cell Profiling

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Staining was performed on single cell suspensions of spleen, thymus, tumor, and mouse peripheral blood. Tissues were harvested and mechanically homogenized and filtered in preparation for staining. Cells were stained for viability using Zombie Red (BioLegend), Zombie NIR (BioLegend) or Fixable Viability Dye 780 (Invitrogen). Fc blocking was performed by incubating cells with Fc Block (anti-CD16/32 clone 93) for 15 minutes followed by a surface stain of fluorochrome-conjugated monoclonal antibodies (a list is provided below) in staining buffer (phosphate-buffered saline + 3% Nu serum). For stains involving intracellular markers cells were permeabilized using Cytofix/Cytoperm (BD Biosciences) for 20 min and washed prior to use of intracellular antibodies or was performed with a FOXP3/transcription factor fixation/permeabilization kit (eBiosciences) according to the manufacturer’s specification. Antibodies: PB-anti-CD45 (30-F11), PB-anti-CD44 (IM7), APC-Cy7-anti-CD45 (30-F11), BV711-anti-CD4 (RM4–5), BV785-anti-CD3 (17A2), BV605-anti-CD8a (53–6.7), Alexa Fluor 700-anti-Ki67 (16A8), FITC-anti-PD-1 (RMP1–30), PE-anti-Tim3 (RMT3–23), PerCP-eFluor 710-anti-Lag3 (eBioC9B7W), and PE-Cy7-anti-CD62L (MEL-14). Additional antibody clones used include: CD45 (clone I3/2.3), CD4 (L3T4), CD8 (53-6.7), CD25 (PC61), PD-1 (29F.1A12) FoxP3(150D/E4) and Ki67 (11F6).

Vick L.V., Collins C.P., Khuat L.T., Wang Z., Dunai C., Aguilar E.G., Stoffel K., Yendamuri S., Smith R J.r., Mukherjee S., Barbi J., Canter R.J., Monjazeb A.M, & Murphy W.J. (2023). Aging augments obesity-induced thymic involution and peripheral T cell exhaustion altering the “obesity paradox”. Frontiers in Immunology, 13, 1012016.

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Quantifying NFAT Nuclear Localization

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Spleen and LN cells from control, CnB ΔTreg or CD28 ΔTreg mice before onset of overt autoimmunity were kept on ice immediately upon harvest, during enrichment of Treg using the Miltenyi mouse Treg selection kit, and dead cell staining with ZombieRed (Biolegend). Some samples were stimulated with ionomycin (1 µg/ml, Sigma) for 10 min. at 37°C, while others were kept on ice to preserve their in situ NFAT activation state. All samples were allowed to adhere to poly-L-lysine coated cover glasses for 5 min at 37°C, then fixed in 4% PFA for 10 min. and stained with anti-NFAT1 (D43B1, Cell Signaling), anti-NFAT2 (7A6, Biolegend), and anti-Foxp3 (FJK-16s, eBioscience) Abs. Primary Ab binding was revealed using Alexa Fluor 633-conjugated anti-rabbit IgG, Alexa Fluor 647-conjugated anti-mouse IgG Fab’ (Jackson Immunoresearch), and Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen), respectively. Slides were mounted using ProLong antifade medium (Thermo Fisher) and analyzed on a Zeiss LSM510 confocal microscope. Nuclear localization of NFAT proteins was quantified as the signaling index of individual cells (30 (link)), which has a value of 0 for full cytoplasmic localization and 1 for complete nuclear localization.

Marangoni F., Zhang R., Mani V., Thelen M., Ali Akbar N.J., Warner R.D., Äijö T., Zappulli V., Martinez G.J., Turka L.A, & Mempel T.R. (2018). Tumor tolerance-promoting function of regulatory T cells is optimized by CD28, but strictly dependent on calcineurin. Journal of immunology (Baltimore, Md. : 1950), 200(10), 3647-3661.

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Flow Cytometric Analysis of Tumor-infiltrating Lymphocytes

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Flow-cytometric analyses of tumor-infiltrating lymphocytes (TILs) and single-cell suspensions from tumors, blood, and lymph nodes were performed using the following antibodies/MHC tetramers. M8 tetramer-PE (H-2Kb, p15E) (from Baylor College of Medicine) with CD8-AF700 (clone KT15/MBL) were used to detect tumor-specific CD8⁺ T cells. CXCR3-APC-Fire750 (clone CXCR3-173) was from Miltenyi Biotec. MHCII-AF700 (clone M5/114.15.2), CD45-BV510 (clone 30-F11), CD3-PE-Cy5.5 or -FITC (clone 145–2 C11), CD49b-FITC (clone DX5), CD19-FITC (clone 1D3), F4/80-FITC (clone 1BM8), CD8-APC (clone 53–6.7), Tim-3-BV605 (clone RMT3-23), Ki-67-BV605 (clone 16A8), CD11c-BV650 (clone N418), CD11b-PE-Cy7 (clone M1/70), CD86-BV421 (clone GL1), Ly6C-PerCP-Cy5.5 (clone HK1.4) and CD103-PE-CF594 (clone M290) were from BioLegend. Other antibodies used were: CD101-Pe-Cy7 (clone Moushi101) from Invitrogen; Phospho-Histone H2A.X-AF488 (#9719) and Isotype Control-AF488 (#2975) were from Cell signaling. 7-AAD (BD Bioscience), Zombie Red, and Zombie NIR (BioLegend) were used to exclude dead cells. The samples were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter). Data were analyzed by using FlowJo software (V.10.4.0; RRID: SCR_008520).

Wang L., Luo R., Onyshchenko K., Rao X., Wang M., Menz B., Gaedicke S., Grosu A.L., Firat E, & Niedermann G. (2023). Adding liposomal doxorubicin enhances the abscopal effect induced by radiation/αPD1 therapy depending on tumor cell mitochondrial DNA and cGAS/STING. Journal for Immunotherapy of Cancer, 11(8), e006235.

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CFSE-Based Proliferation Assay for Memory CD4+ T Cells

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Memory CD4+ T cells from the blood of two HIV-seronegative donors were purified as described above, and then resuspended at 2 × 10⁷ cells/mL in PBS containing 0.1% FBS. The cells were then loaded for 8 min with 1.5 μM of the proliferation dye CFSE (ThermoFisher). Labeling was then stopped by the addition of an equal volume of pre-warmed, 100% FBS, and the labeled cells were incubated at 37°C for an additional 10 min. The sample was then washed three times in RP10. Memory CD4+ T cells not exposed to CFSE were treated identically in parallel. The time = 0 specimen was immediately stained as described below. The remaining cells were cultured with or without activation with 16 nM PMA, 1 μM ionomycin, and 100 IU/mL IL2 (all three reagents from Thermofisher). At the indicated time points, cells were stained with APC/Cyanine7 anti-human CD3 (Clone SK7, Biolegend), PE/Cy7 anti-human CD4 (Clone A161A1, Biolegend), Alexa Fluor 700 anti-human CD8 (Clone SK1, Biolegend), and Zombie Red (Biolegend) as a Live/Dead discriminator. Stained cells were fixed and analyzed by FACS on an LSRII (BD Biosciences). Flowjo (BD Biosciences) was used for analysis. Live, singlet CD3+CD4+CD8- cells were assessed for proliferation by monitoring the loss of CFSE signal. Results shown are from one of two donors which gave similar results.

Neidleman J., Luo X., Frouard J., Xie G., Hsiao F., Ma T., Morcilla V., Lee A., Telwatte S., Thomas R., Tamaki W., Wheeler B., Hoh R., Somsouk M., Vohra P., Milush J., James K.S., Archin N.M., Hunt P.W., Deeks S.G., Yukl S.A., Palmer S., Greene W.C, & Roan N.R. (2020). Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir. eLife, 9, e60933.

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Dissociated Fibroadenoma Cell Phenotyping

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The dissociated single cells from primary fibroadenoma tissues or normal tissues were resuspended in 100 μl of cell staining buffer (Biolegend, Cat.# 420201) and stained with Zombie Red™ (Biolegend, Cat.# 423109) to distinguish live cells for 30 min at room temperature according to the manufacturer’s instructions. Then, the cells were pelleted at 400 × g for 10 min at 4 °C, resuspended in 100 μl of cell staining buffer and stained with a series of FACS antibodies, including CD45 (Biolegend, clone: HI30, cat. # 304014, [1:100]), CD31 (Biolegend, clone: WM59, cat. # 303118, [1:100]), EpCAM (Biolegend, clone: 9C4, cat. # 324204, [1:100]) and CD49f (Biolegend, clone: GoH3, cat. # 313612, [1:100]). Staining was performed for 30 min with FACS antibodies on ice and washed twice with processing buffer (PBS, 5% BSA). After washing, the cells were resuspended in 200–300 μL of cell staining buffer and analyzed on a FACSCanto II flow cytometer (BD Biosciences). Cell sorting was performed on a FACSAria III cell sorter (BD Biosciences). Data were analyzed with FlowJo software (V10.0 for Windows).

Chen Z., Zhang Y., Li W., Gao C., Huang F., Cheng L., Jin M., Xu X, & Huang J. (2023). Single cell profiling of female breast fibroadenoma reveals distinct epithelial cell compositions and therapeutic targets. Nature Communications, 14, 3469.

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Multiparametric Flow Cytometry of Cervical Cancer Tissue

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Fresh biopsy of cervical cancer tissue was washed with cold RPMI 1640 medium, then the tissue was minced using razor blades and mechanically homogenized in cold RPMI 1640 medium containing 2.5% fetal bovine serum [20 (link)]. The suspensions were filtered using a 70μm cell strainer (BD Biosciences) and samples were stained immediately for phenotype analysis by flow cytometry. All steps were completed within 2 hours. Cell surface marker and intracellular cytokine staining for flow cytometry analysis were performed as described previously [21 (link), 22 ]. Live/dead discrimination was performed with fixable viability dye (Zombie Red, BioLegend). The fluorescent antibodies used in flow cytometry analysis were listed in Table 1.Table 1

The fluorescent antibodies used in flow cytometry analysis

Antibody	Clone (source)	Isotype
CD163	GHI/61 (Biolegend)	IgG1
IL-10	JES3-9D7 (Biolegend)	IgG1
PD-1	NAT105 (Biolegend)	IgG1
CD68	FA-11 (Biolegend)	IgG2a
CD14	63D3 (Biolegend)	IgG1
CD45	HI30 (Biolegend)	IgG1
CCR7	150503 (BD Biosciences)	IgG2a
iNOS	Clone 6 (BD Biosciences)	IgG2a
CD9	M-L13 (BD Biosciences)	IgG1
TNFα	MAb11 (BD Biosciences)	IgG1
CD11b	M1/70 (BD Biosciences)	IgG2b

Ren J., Li L., Yu B., Xu E., Sun N., Li X., Xing Z., Han X., Cui Y., Wang X., Zhang X, & Wang G. (2022). Extracellular vesicles mediated proinflammatory macrophage phenotype induced by radiotherapy in cervical cancer. BMC Cancer, 22, 88.

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Multiparameter Flow Cytometry of Mouse Immune Cells

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Isolated immune cells were washed and blocked with αCD16/CD32 (100ug/100,000 cells, #101320, BioLegend) for 5 minutes on ice and then stained with labeled antibodies against surface markers or cytokines on ice for 30 minutes in FACS buffer (2% FCS/PBS). Intracellular staining for Foxp3 was performed using a Foxp3/Transcription Factor Staining Kit (#00-5523, eBioscience) following surface marker staining and was used according to manufacturer’s instructions. The following monoclonal antibodies directed against mouse antigens were used for flow cytometry: BioLegend - CD45 (clone 30-F11), CD3 (17A2), CD4 (GK1.5), Foxp3 (FJK-16S), CD8 (53-6.7), NK1.1 (PK136), CD11c (N418), CD19 (6D5), CD5 (53-7.3), CD21/35 (7E9), IgD (11–26), IgM (RMM-1), CD23 (B3B4), CD138 (281-2); BD Biosciences - CD1d (clone 1B1), CD93 (AA4.1), CD11b (M1/70). Viability of cells was determined by staining with either Zombie NIR (#423105, BioLegend) or Zombie Red (#423113, BioLegend) viability dye. All samples were acquired on LSRII Fortessa (BD Bioscience) at UNC Flow Cytometry Core Facility and analyzed by FlowJo version 10.2 (TreeStar, Inc.).

Michaud D., Mirlekar B., Steward C., Bishop G, & Pylayeva-Gupta Y. (2022). B Cell Receptor Signaling and Protein Kinase D2 Support Regulatory B Cell Function in Pancreatic Cancer. Frontiers in Immunology, 12, 745873.

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