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Zombie violet viability dye

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Zombie Violet viability dye is a fluorescent dye used to detect dead cells in flow cytometry analysis. It binds to nucleic acids of cells with compromised cell membranes, allowing the identification of non-viable cells.

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Lab products found in correlation

Facsaria 3, by BD (2 mentions) Anti foxp3 fjk 16a, by Thermo Fisher Scientific (1 mentions) Anti cd73 ty 11.8, by BioLegend (1 mentions) Fitc annexin 5 staining kit, by BioLegend (1 mentions) Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Aria 3, by BD (1 mentions) Facscalibur, by BD (1 mentions) Human fc block, by BioLegend (1 mentions) Rnasin plus rnase inhibitor, by Promega (1 mentions) Facsaria cell sorter, by BD (1 mentions) Ic fixation buffer kit, by Thermo Fisher Scientific (1 mentions) Software, by FlowJo (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Cd45 fitc, by BioLegend (1 mentions) Cd31 pe, by BD (1 mentions) Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions) Cd11b m1 70, by BioLegend (1 mentions) Mouse fc block, by BD (1 mentions) Collagenase 1, by Thermo Fisher Scientific (1 mentions) Macsquant, by Miltenyi Biotec (1 mentions)

8 protocols using zombie violet viability dye

1

Multiparameter Flow Cytometry Analysis

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Anti-CD4 (GK1.5), anti-CD25 (PC-61), anti-Vα3.2 (RR3-16), anti-CD45.2 (104), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-ICOS (C398.4A), anti-PD-1 (29F.1A12) and anti-CD73 (TY/11.8) were from BioLegend. Anti-Foxp3 (FJK-16a) was from eBioscience. Cell sorting and analyses were performed on ARIA III, FACS CALIBUR, LSRII, and CANTO (BD). For detection of apoptosis, FITC Annexin V staining kit and Zombie Violet viability dye were used from BioLegend. For intracellular staining cells were fixed and permeabilized using Fixation/Permeabilization buffers from eBioscience and BD according to manufacturers’ manual.
Jones A., Opejin A., Henderson J.G., Gross C., Jain R., Epstein J.A., Flavell R.A, & Hawiger D. (2015). Peripherally induced tolerance depends on pTreg cells that require Hopx to inhibit intrinsic IL-2 expression. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1489-1497.
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2

ZIKV-Infected Macrophage Isolation

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Macrophages were washed once with 1× PBS, gently scraped off in 1× PBS, pelleted at 4 °C, and stained with Zombie Violet viability dye (BioLegend) in the dark. Cells were washed once with FACS buffer (1× PBS, 3% FBS, and 2 mM EDTA) and were fixed and permeabilized with 4% paraformaldehyde (15710-S; Electron Microscopy Sciences) and 0.1% saponin (47036; Sigma-Aldrich) in molecular-grade PBS supplemented with 1:100 RNasin Plus RNase Inhibitor (N2615; Promega) for 30 min at 4 °C. Cells were washed in wash buffer (1× PBS containing 0.2% BSA, 0.1% saponin, and 1:100 RNasin Plus RNase Inhibitor) and were blocked for 10 min with human Fc Block (1:500) (BioLegend) in staining buffer (1× PBS containing 1% BSA, 0.1% saponin, and RNasin Plus RNase Inhibitor). HMDMs were then stained with 4G2 antibody (BioXcell) conjugated to AF647 (catalog no. A20186; Thermo Scientific) for 30 min at 4 °C, washed twice, resuspended at 5–10E6 cells/mL in sort buffer (PBS containing 0.5% BSA and 1:25 RNasin Plus RNase Inhibitor) and sorted into ZIKV+ and ZIKV− cells on a FACSAria cell sorter (BD Biosciences) at the La Jolla Institute for Allergy and Immunology. Gates were set with reference to negative controls.
Carlin A.F., Vizcarra E.A., Branche E., Viramontes K.M., Suarez-Amaran L., Ley K., Heinz S., Benner C., Shresta S, & Glass C.K. (2018). Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages. Proceedings of the National Academy of Sciences of the United States of America, 115(39), E9172-E9181.
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3

SULT2B1b Knockdown in LNCaP Cells

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Forty-eight hours following siRNA transfection, LNCaP cells were harvested with trypsin and stained with Zombie Violet viability dye (Biolegend, 423114) before sorting of viable cells on a FACS ARIA III (BD Biosciences). Cells were loaded and stained in a 17–25 µm IFC plate (Fluidigm, 100–5761) followed by exclusion of capture sites containing doublets or dead cells. This procedure was completed on consecutive days for Control and SULT2B1b KD cells and repeated three times for biological replicates. In total, 209 Control siRNA and 190 SULT2B1b siRNA treated cells were used for analysis. Additional details regarding C1 loading and subsequent library preparation is located in Supplemental Methods.
Vickman R.E., Yang J., Lanman N.A., Cresswell G.M., Zheng F., Zhang C., Doerge R.W., Crist S.A., Mesecar A.D., Hu C.D, & Ratliff T.L. (2019). Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNF alpha in Castration Resistant Prostate Cancer. Molecular cancer research : MCR, 17(6), 1253-1263.
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4

Flow Cytometric Analysis of Immune Cells

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A total of 1–2 × 106 cells were stained with Zombie Violet viability dye (Biolegend, San Diego, CA, USA) according to the manufacturer specifications and then washed with 500 μL FACS buffer (PBS, 2% FBS, 0.1% sodium azide) before a 10 min incubation with 0.5 µg FcR blocking antibody (2.4G2). The cells were washed with FACS buffer and incubated with a surface antibody cocktail for 15 min on ice. The cells were washed twice before staining intracellularly for MPO using the IC fixation buffer kit from eBioscience (San Diego, CA, USA) in accordance with manufacturer instructions. The cells were re-suspended into FACS buffer and data were acquired from the live cells on a BD LSRII flow cytometer. The data were analyzed with FlowJo software (FlowJo, Ashton, OR, USA).
Gurski C.J, & Dittel B.N. (2022). Myeloperoxidase as a Marker to Differentiate Mouse Monocyte/Macrophage Subsets. International Journal of Molecular Sciences, 23(15), 8246.
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5

Immunophenotyping of Dermal Fibroblasts

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Freshly isolated dFB from rat skin was stained with zombie violet viability dye (Biolegend, 423114) to stain dead cells. Cells were then stained with an antibody cocktails containing PE-CD31 (BD, 555027), FITC-CD45 (BioLegend, 202205), PerCP/Cy5.5-CD29 (BioLegend, 102227), and PE-Cy7-CD34 (SantaCruze, sc-7324). FACS analysis for protein expression of each cell marker was performed by the BD FACSCanto RUO machine and analyzed by FlowJo V10 software.
Chen L., You Q., Liu M., Li S., Wu Z., Hu J., Ma Y., Xia L., Zhou Y., Xu N, & Zhang S. (2022). Remodeling of dermal adipose tissue alleviates cutaneous toxicity induced by anti-EGFR therapy. eLife, 11, e72443.
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6

Isolation of CD98+ Monocytes from Parasitic Infection

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2 × 106 monocytes from three donors were separately seeded in 6-well cell culture plates and allowed to settle overnight. Freshly prepared parasites were then added to the cells at a MOI of 1, briefly centrifuged, and incubated for an additional 1 h at 37 °C with 5% CO2. The cells were collected, washed once with PBS, resuspended in blocking buffer (PBS supplemented with 1% Goat serum) and incubated on ice for 30 min. The cells were centrifuged at 400 × g for 4 min at 4 °C and washed once in ice-cold FACS buffer. CD98+ were sorted in BD FACSAria III (BD Biosciences) after double staining with Alexa Flour 488 anti-human CD98 (Novus Biologicals) monoclonal antibodies and Zombie Violet viability dye (Biolegend).
Patir A., Gossner A., Ramachandran P., Alves J., Freeman T.C., Henderson N.C., Watson M, & Hassan M.A. (2020). Single-cell RNA-seq reveals CD16- monocytes as key regulators of human monocyte transcriptional response to Toxoplasma. Scientific Reports, 10, 21047.
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7

Neutrophil CD11b surface activation

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Primary human neutrophils (3x10 6 ) were suspended in Dulbecco's PBS without calcium or magnesium (Gibco) with 0.1% glucose (DPBSG-). Neutrophils were left untreated or exposed to 200 nM PMA for 10 min. After treatment with blocking antibodies or isotype control (20 mg ml -1 ), unlabeled or CFSE-labeled Ngo was added to the neutrophils at multiplicity of infection = 1 unless otherwise indicated and incubated at 37 o C for 1 hr with slow rotation. Cells were stained with Zombie Violet viability dye (BioLegend), fixed with 1% paraformaldehyde (PFA) for 10 min, and analyzed by imaging flow cytometry as above. Surface presentation of total (ICRF44) and active (CBRM1/5) CD11b was analyzed by flow cytometry.
Smirnov A., Daily K.P., Gray M.C., Ragland S.A., Werner L.M., Brittany Johnson M., Eby J.C., Hewlett E.L., Taylor R.P, & Criss A.K. (2023). Phagocytosis via complement receptor 3 enables microbes to evade killing by neutrophils. Journal of leukocyte biology, 114(1).
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8

Comprehensive Immune Cell Profiling

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For each mouse, a part of the spleen was mechanically dissociated while one of each of the paired lobes of the prostate was digested in 1mg/mL collagenase I from Life Technologies, (Carlsband, CA) for up to one hour while shaking at 37° C. After digestion/dissociation, cell samples were washed and filtered before RBC removal. Cells were blocked with mouse Fc block (BD Pharmingen, 553142) and stained with Zombie Violet viability dye (Biolegend) followed by staining with antibodies to CD45 [30-F11], CD4[GK1.5], CD8[53–6.7], CD19[6D5], F4/80 [BM8], and CD11b[M1/70] according to the manufacturer’s instructions (Biolegend). All samples were washed and filtered before flow analysis on a MACSQuant (Miltenyi). Final data analysis was conducted using FlowLogic software version 700.2A (Miltenyi). All cell events underwent debris and doublet exclusion followed by removal of dead cells containing Zombie Violet dye. The CD45+ cells were gated on the viable cell population and the specific immune cell subsets were gated on the CD45+ cell population. Three mice were analyzed for each group, where each point represents a single mouse.
Aaron-Brooks L.M., Sasaki T., Vickman R.E., Wei L., Franco O.E., Ji Y., Crawford S.E, & Hayward S.W. (2019). Hyperglycemia and T-Cell infiltration are associated with stromal and epithelial prostatic hyperplasia in the Non-obese diabetic (NOD) mouse.. The Prostate, 79(9), 980-993.
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