Live dead fixable violet

Manufactured by Thermo Fisher Scientific

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The LIVE/DEAD Fixable Violet is a fluorescent dye used for identifying and enumerating live and dead cells in flow cytometry analysis. The dye binds to cellular proteins, allowing for the discrimination between viable and non-viable cells.

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LIVE/DEAD Fixable Violet Dead Cell Stain Kit (172 citations) LIVE/DEAD Violet (58 citations) LIVE/DEAD Fixable Violet stain (34 citations) LIVE/DEAD Fixable Violet Dead Cell staining kit (6 citations) LIVE/DEAD Fixable Violet Dead cell dye (5 citations) Fixable live/dead (2 citations) Live/Dead violet fluorescence fixable dead cell stain reactive kit (2 citations) Live/dead ™ Fixable Violet Dead Cells Stain Kit (2 citations) LIVE/DEAD Fixable Violet Dead Cell Stain Sampler Kit (2 citations) Live/Dead Fixable Aqua or Violet Stain (1 citations)

41 protocols using live dead fixable violet

Isolation and Analysis of Murine Muscle Cells

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Both TA muscles from 1 mouse were pooled, finely minced, and digested for 60 minutes at 37°C for in Hanks’ balanced salt solution lacking calcium and magnesium (HBSS^–/–, Thermo Fisher Scientific), supplemented with 250 U/mL collagenase II (Thermo Fisher Scientific), 4.0 U/mL dispase (MilliporeSigma), and 2.5 mmol/L CaCl₂ (MilliporeSigma). The resulting digest solution was filtered through a 40 μm cell strainer and centrifuged at 350g for 5 minutes. Cells were Fc blocked with a CD16/CD32 antibody (Thermo Fisher Scientific, 14-0161-82) for 10 minutes at 4°C and then incubated for 30 minutes on ice with primary antibodies, including including CD45-PE (30-F11) (Thermo Fisher Scientific, 12-0451-82), Ly-6G-FITC (1A8) (BD Pharmingen, 551460), CD64-APC (X54-5/7.1) (BioLegend, 139306, CD11c-APCe780 (N418) (BioLegend, 47-0114-80), and Live/Dead fixable violet (Thermo Fisher Scientific, L34963). Flow cytometry was performed using LSRFortessa cell analyzer (BD Biosciences) and data were analyzed with FlowJo software.

Markworth J.F., Brown L.A., Lim E., Floyd C., Larouche J., Castor-Macias J.A., Sugg K.B., Sarver D.C., Macpherson P.C., Davis C., Aguilar C.A., Maddipati K.R, & Brooks S.V. (2020). Resolvin D1 supports skeletal myofiber regeneration via actions on myeloid and muscle stem cells. JCI Insight, 5(18), e137713.

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Quantifying Intracellular ROS in Myeloid Cells

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Ly6C⁺ cells were isolated from in vitro moDC and M-MDSC cultures and plated at 6x10⁴ cells per well in a 96 nonpyrogenic flat bottom plate where some wells were coated with active CD47 protein. In certain conditions, cells were pre-treated with anti-SIRPα blocking antibody as described above. The cells were incubated for 4h in GM-CSF alone, GM-CSF-TCM, GM-CSF-TCM-activeCD47 or anti-SIRPα-GM-CSF-TCM-activeCD47. After incubation, cells were washed and treated with 10μM 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA; Sigma-Aldrich) in basal DMEM for 25min. Immediately after, cells were washed twice with PBS and transferred to ice. Then, they were resuspended in PBS with Live/Dead Fixable Violet (Thermo, Cat: 62248) viability dye, diluted 1:1000, for 3min, to label dead cells. Samples were Immediately washed once with PBS and run on an LSR Fortessa cell analyzer (BD, Biosciences) to measure DCFDA levels as a readout for intracellular ROS.

Zimarino C., Moody W., Davidson S.E., Munir H, & Shields J.D. (2024). Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells. iScience, 27(4), 109546.

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CD8+ T Cell Cytolytic Assay

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P815 mastocytoma target cells were labeled with LIVE/DEAD Fixable Violet (Thermo Fisher Scientific) and TFL4 (OncoImmun), washed twice in PBS, and incubated for 30 min at room temperature with α-CD3 (1 μg/mL; clone UCHT1; Bio-Rad). CD8⁺ T cells were negatively selected from blood and TDL using a CD8⁺ T Cell Enrichment Kit (StemCell Technologies). Isolated CD8⁺ T cells were rested in complete medium for at least 45 min at 37°C and then incubated with α-CD3-coated P815 cells at different E:T ratios in a 96-well V-bottom plate for 4 hr at 37°C. Cells were then stained with α-active caspase-3–FITC (clone C92–605; BD Biosciences) and α-CD3–PE (clone SK7; BD Biosciences) and acquired using an LSRII (BD Biosciences). Cytolytic activity was calculated by subtracting the frequency of active caspase-3⁺TFL4⁺LIVE/DEAD⁻ P815 cells in target-only wells from the frequency of active caspase-3⁺TFL4⁺LIVE/DEAD⁻ P815 cells in wells containing CD8⁺ T cells.

Buggert M., Vella L.A., Nguyen S., Wu V., Chen Z., Sekine T., Perez-Potti A., Maldini C.R., Manne S., Darko S., Ransier A., Kuri-Cervantes L., Japp A.S., Brody I.B., Ivarsson M.A., Gorin J.B., Rivera-Ballesteros O., Hertwig L., Antel J.P., Johnson M.E., Okoye A., Picker L., Vahedi G., Sparrelid E., Llewellyn-Lacey S., Gostick E., Sandberg J., Björkström N., Bar-Or A., Dori Y., Naji A., Canaday D.H., Laufer T.M., Wells A.D., Price D.A., Frank I., Douek D.C., Wherry E.J., Itkin M.G, & Betts M.R. (2020). The identity of human tissue-emigrant CD8+ T cells. Cell, 183(7), 1946-1961.e15.

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Multicolor Flow Cytometry Staining

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Samples were resuspended in PBS with Live/Dead Fixable Violet (Thermo, Cat: 62248) viability dye, diluted 1:1000, for 15min. After washing, samples were incubated with fluorophore-conjugated primary antibodies prepared at 1:300 dilution in FACS buffer (0.5% BSA in PBS) and mixed 1:1 with Fc block (generated in house from a rat 2.4G2 hybridoma cell line)), for 40min, at 4°C. After surface staining, and if intracellular epitope detection was required, samples were fixed, permeabilised and stained in accordance with the FoxP3/ Transcription Factor Staining Kit (eBioscience, Cat: 00- 5523). Briefly, cells were incubated with fluorophore-conjugated primary antibodies, diluted 1:300 in permeabilization buffer, for 30min at RT. After washing, samples were run on an LSR Fortessa cell analyzer (BD Biosciences) and analysed using FlowJo version 10. (FlowJo, BD Biosciences).

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Flow Cytometry Analysis Protocol

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Flow cytometric analyses were performed using the 13-color CytoFLEX S (Beckman Coulter) instrument to acquire data. Data were analyzed using FlowJo v10.8 (Becton Dickenson and Company). DAPI (0.5 mg/mL, Thermo Fisher Scientific, cat. no. D1306) or a live/dead fixable violet (Thermo Fisher Scientific, cat. no. L34964) was used to exclude dead cells in all experiments. Antibodies to the murine and human proteins used for flow cytometry are listed in Supplementary Table S1.

Drakes D.J., Abbas A.M., Shields J., Steinbuck M.P., Jakubowski A., Seenappa L.M., Haqq C.M, & DeMuth P.C. (2024). Lymph Node–Targeted Vaccine Boosting of TCR T-cell Therapy Enhances Antitumor Function and Eradicates Solid Tumors. Cancer Immunology Research, 12(2), 214-231.

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Dual Fluorescent Reporter Assay

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The FUW mCherry-GFP-LC3 reporter plasmid was obtained from Addgene (Plasmid #110060) which was utilized to generate lentivirus in 293 T cells. B16-F10 Cas9-expressing cells were then infected and grown for 48 h. Cells were then plated and treated with standard DMEM with 10% FBS or Hanks Balanced Salt Solution for the indicated time. Following the time course all cells were washed 3x with PBS and then resuspended in 100 uL of PBS and stained with LIVE/DEAD Fixable Violet (ThermoFisher L34955) stain per the manufacturer’s instructions.
Cells were then passed through a 40-micron filter to generate a single cell suspension and then run on a BD FACSCelesta. Single cells were gated for live cells and then the individual GFP and mCherry signal were measured in the FITC and CF594 channels, respectively. Data were analyzed in FlowJo v10.

Gabel A.M., Belleville A.E., Thomas J.D., McKellar S.A., Nicholas T.R., Banjo T., Crosse E.I, & Bradley R.K. (2024). Multiplexed screening reveals how cancer-specific alternative polyadenylation shapes tumor growth in vivo. Nature Communications, 15, 959.

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HLA-B*27:05 Peptide Dextramer Staining

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PBMCs were stained with LIVE/DEAD Fixable Violet (Thermo Fisher Scientific). Cells were then washed twice and incubated with HLA-B^∗27:05-peptide dextramers as per the manufacturer's protocol (Immudex). Surface markers were identified using anti-CD3–ECD (Beckman Coulter), anti-CD4–APC-eFluor780 (eBioscience), and anti-CD8–PerCP (BD Biosciences). Cells were then washed again and fixed with 1% formaldehyde for a minimum of 1 h. Non-identical HLA-A^∗A2:01-peptide dextramers containing an irrelevant epitope were used as gating controls. Unstained and single-stained cells were used to determine compensation values. Samples were acquired using a CyAn ADP Analyzer (Beckman Dako Cytomation), and data were analyzed using FlowJo software (Tree Star).

Pymm P., Tenzer S., Wee E., Weimershaus M., Burgevin A., Kollnberger S., Gerstoft J., Josephs T.M., Ladell K., McLaren J.E., Appay V., Price D.A., Fugger L., Bell J.I., Schild H., van Endert P., Harkiolaki M, & Iversen A.K. (2022). Epitope length variants balance protective immune responses and viral escape in HIV-1 infection. Cell Reports, 38(9), 110449.

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Phenotyping Immune Cell Surface Markers

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To study cell-surface markers, cells were collected using Versene, a non-enzymatic dissociation buffer (ThermoFisher). Cells were resuspended in the staining buffer (PBS with 4% fetal bovine serum and 2 mM ethylenediaminetetraacetic acid (EDTA)). Cells were then incubated in ice with Fc block reagent (Miltenyi Biotec) for 10 minutes, and stained with the viability dye LIVE/DEAD™ Fixable Violet (ThermoFisher), following the manufacturer's protocol.
Cells were then stained to study the proteins of interest, using the following antibodies: CD16 (APC) (#130-113-389, Miltenyi Biotec), CD14 (APC) (#130-110-520, Miltenyi Biotec), CD163 (FITC) (#33618, BioLegend), CD1a (PE) (#300106, BioLegend), CD80 (PE) (#H12208P, eBioScience), CD83 (APC) (#130-110-504, Miltenyi Biotec), CD86 (APC) (#130-113-569, Miltenyi Biotec), HLA-DR (PE) (#12-9956-42, eBioScience).
After staining, cells were fixed with PBS + 4% paraformaldehyde (Electron Microscopy Sciences) and analyzed within 2 days using a BD FACSCanto™ II Cell Analyzer (BD Biosciences). Data were analyzed with the FlowJo v10 software.

Morante-Palacios O., Ciudad L., Micheroli R., de la Calle-Fabregat C., Li T., Barbisan G., Houtman M., Edalat S.G., Frank-Bertoncelj M., Ospelt C, & Ballestar E. (2021). Coordinated glucocorticoid receptor and MAFB action induces tolerogenesis and epigenome remodeling in dendritic cells. Nucleic Acids Research, 50(1), 108-126.

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Isolation of Neutrophils from Whole Blood

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Whole blood was collected from healthy donors between age 18–65 in accordance with the Declaration of Helsinki guidelines and the Institutional Review Board of the University of California Irvine. Red blood cells were separated in 3% Dextran (Sigma Aldrich, St. Louis, MO) PBS and neutrophils were purified from remaining cells by overlay on a Ficoll (GE Healthcare) density gradient and centrifugation at 500xg for 25 min. Remaining RBCs were lysed and neutrophils were re-suspended in RPMI 1640. Purity (>90%) was assessed by flow cytometry using anti-human CD16 and CD66b antibodies (eBioscience, San Diego, CA). Live/Dead fixable violet (Thermo Fisher) was used to gate live cells and positive populations were identified using FMO controls.

Clark H.L., Jhingran A., Sun Y., Vareechon C., Carrion S.D., Skaar E.P., Chazin W.J., Calera J.A., Hohl T.M, & Pearlman E. (2015). Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection. Journal of immunology (Baltimore, Md. : 1950), 196(1), 336-344.

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Single-Cell Immune Profiling of Infection

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Only day 9 post-infection samples were sorted for scRNA-seq to obtain a roughly equal proportion of immune and non-immune cells for analysis, although all samples were stained to examine the relative proportions of immune and non-immune cells. Single-cell suspensions were incubated with biotin-conjugated antibodies targeting the following Lineage markers for 30 min at 4 °C in 10% RPMI: CD3e, CD8a, TCRb, B220, NK1.1, Ly6C/G (anti-GR1), CD11b, CD11c, Ter119 (Biolegend). Samples were then washed twice in 10% RPMI and stained with Live/Dead Fixable Violet (ThermoFisher), streptavidin, and anti-CD90 for 30 min at 4 °C in 10% RPMI. Samples were then run on an Aria IIIu cell sorter (BD Biosciences).

Kasmani M.Y., Topchyan P., Brown A.K., Brown R.J., Wu X., Chen Y., Khatun A., Alson D., Wu Y., Burns R., Lin C.W., Kudek M.R., Sun J, & Cui W. (2023). A spatial sequencing atlas of age-induced changes in the lung during influenza infection. Nature Communications, 14, 6597.

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