Live dead fixable blue dead cell stain

Manufactured by Thermo Fisher Scientific

Sourced in United States

The LIVE/DEAD Fixable Blue Dead Cell Stain is a fluorescent dye used to detect dead cells in a cell population. It is designed to bind to proteins in dead cells, allowing for their identification and exclusion from analysis.

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Lab products found in correlation

Lsrii flow cytometer, by BD (6 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (6 mentions) Propidium iodide, by Thermo Fisher Scientific (6 mentions) Anti cd45, by BioLegend (5 mentions) Facsaria 2, by BD (4 mentions) Lsrfortessa, by BD (4 mentions) Lsr 2, by BD (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Brefeldin a, by Merck Group (3 mentions) Facsdiva software, by BD (3 mentions) Human trustain fcx, by BioLegend (3 mentions) Anti foxp3 fjk 16s, by Thermo Fisher Scientific (3 mentions) Collagenase, by Merck Group (2 mentions) Bdca 1, by Thermo Fisher Scientific (2 mentions) Bdca 2, by Thermo Fisher Scientific (2 mentions) Bdca 3, by Thermo Fisher Scientific (2 mentions) Anti human cd34 pe cy7, by BioLegend (2 mentions) Cd45ra bv 421, by BD (2 mentions) Cd38 apc hit2, by BD (2 mentions) Cd90 bv605, by BioLegend (2 mentions)

Close spelling variants of this product

LIVE/DEAD stain (166 citations) Live/Dead Blue (66 citations) Fixable live/dead stain (61 citations) LIVE/DEAD Fixable Blue (45 citations) LIVE/DEAD® fixable blue stain (19 citations) Live/dead blue stain (5 citations) Fixable live/dead (2 citations) Live/Dead Fixable Blue Dead Cells Stain Kit (2 citations)

74 protocols using live dead fixable blue dead cell stain

Induction of RICD in Effector T Cells

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RICD was induced in day 6 or 7 effector T cells by incubation on plate-bound anti-CD3 (10 μg/mL) for 16–18 h at 37 °C. Cells were removed and stained with Live/Dead Fixable Blue Dead Cell Stain (ThermoFisher Scientific), fixed in 1% formaldehyde (v/v), and analyzed by flow cytometry.
As a positive control for cell death, day 6 effector T cells were incubated with 400 ng/mL FLAG-tagged FasL (Enzo Life Sciences, Farmingdale, NY, USA) and 2 μg/mL anti-FLAG antibody M-2 (Sigma-Aldrich) for 1.5 h at 37 °C. Cells were then stained with Live/Dead Fixable Blue Dead Cell Stain (ThermoFisher Scientific) fixed in 1% formaldehyde (v/v) and analyzed by flow cytometry.

Secinaro M.A., Fortner K.A., Dienz O., Logan A., Murphy M.P., Anathy V., Boyson J.E, & Budd R.C. (2018). Glycolysis promotes caspase-3 activation in lipid rafts in T cells. Cell Death & Disease, 9(2), 62.

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Characterization of Human Nasal Dendritic Cells

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The human nasopharyngeal samples were processed immediately post-surgery. The tissue was minced, treated with 0.25 mg/ml of collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 60 minutes and cell isolation was performed. Cells were surface stained with CD45, CD20, CD3, CD335, CD66b, BDCA-1, BDCA-2, BDCA-3, CD14, and Live/Dead Fixable Blue Dead Cell Stain (Life technologies, Grand Island, NY). Titrated Abs were added to the cells in 50 μl PBS 1% FCS for 30 min at 4°C. Washed cells were fixed in 2% formaldehyde and stored at 4°C until analysis, which was performed using an LSR II flow cytometer (BD Biosciences, San Jose, CA). The whole sample was acquired and analysis was preformed using Flow Jo 9.1 software (Tree Star, Ashland, OR). To identify human DC subsets, dead cells were excluded and CD3⁺, CD20⁺, CD335⁺, CD66b⁺, and CD14⁺ cells i.e., T, B, NK, neutrophils and monocytes were sequentially gated out; three DC populations were identified within the CD45⁺ mononuclear cells by gating respectively on either BDCA-1⁺ cells, BDCA-2⁺ cells or BDCA-3^hi cells.

Lee H., Ruane D., Law K., Ho Y., Garg A., Rahman A., Esterházy D., Cheong C., Goljo E., Sikora A.G., Mucida D., Chen B., Govindraj S., Breton G, & Mehandru S. (2015). Phenotype and function of nasal dendritic cells. Mucosal immunology, 8(5), 1083-1098.

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Isolating and Modifying CD34+ HSPCs

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Harvested wells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies) and then with anti human CD34 PE-Cy7 (581, BioLegend), CD38 Alexa Fluor 647 (AT1, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), CD45RA BV 421 (HI100, BD Biosciences), and CD90 BV605 (5E10, BioLegend) and analyzed by flow cytometry. For sorting of CD34⁺ or CD34⁺/CD38^-/CD90⁺ cells, CB-derived CD34⁺ HSPCs were stained directly after isolation from blood with anti human CD34 FITC (8G12, BD Biosciences), CD90 PE (5E10, BD Biosciences), CD38 APC (HIT2, BD Bioscience), and cells were sorted on a FACS Aria II (BD Bioscience), cultured overnight, and then electroporated with HBB RNP and transduced with HBB GFP rAAV6 using our optimized parameters.

Dever D.P., Bak R.O., Reinisch A., Camarena J., Washington G., Nicolas C.E., Pavel-Dinu M., Saxena N., Wilkens A.B., Mantri S., Uchida N., Hendel A., Narla A., Majeti R., Weinberg K.I, & Porteus M.H. (2016). CRISPR/Cas9 Beta-globin Gene Targeting in Human Hematopoietic Stem Cells. Nature, 539(7629), 384-389.

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Multiparametric Analysis of Immune Cells

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Spleen and draining LNs were harvested and prepared for flow cytometry as was described for cell culture previously. Skin grafts were harvested, minced into approximately 1 mm² pieces, and placed into 2 mL of Hank's buffered salt solution with Liberase (Roche Diagnostics, Indianapolis, IN). After incubation at 37°C for 30 minutes, the digested tissue was passed through a 100-μm cell strainer and washed twice with supplemented RPMI. Splenocytes (SPLs), LN cells, and skin graft cells were stimulated with phorbal 12-myristate 13-acetate (PMA) and ionomycin in the presence of Brefeldin A for 4 hours at 37°C. Cells were stained for 30 minutes at room temperature first with LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies) and then with surface stain antibodies for 20 minutes as indicated in Table S1, SDC,http://links.lww.com/TXD/A37. The cells were then processed for intracellular staining using Intracellular Fixation & Permeabilization Buffer Set (eBioscience) as described by the manufacturer. Cells were then stained with intracellular antibodies (Table S1, SDC,http://links.lww.com/TXD/A37) overnight at 4°C and analyzed using a BD LSR Fortessa (BD Biosciences, San Jose, CA).

Julliard W., Fechner J.H., Owens L., O'Driscoll C.A., Zhou L., Sullivan J.A., Frydrych L., Mueller A, & Mezrich J.D. (2017). Modeling the Effect of the Aryl Hydrocarbon Receptor on Transplant Immunity. Transplantation Direct, 3(5), e157.

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Assessing PBMC Activation by M.tb

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Human PBMC (1 × 10⁶/well) were plated in 96-well plates in 200µl of antibiotic-free RPMI 1640 containing with 10% heat inactivated human AB serum and 5µg/ml brefeldin-A (Sigma-Aldrich, St. Louis, MO) in the absence or presence of 100 µg/ml of γ-irradiated M. tb for 6 hours at 37°C in 5% CO2. Cells were washed and stained with Live/Dead fixable blue dead cell stain (Life Technologies) for 20 minutes at room temperature (RT) then washed with 1% PBS/BSA and stained with antibodies for extracellular surface markers for 1h at RT. Cells were then fixed and permeabilized (Foxp3/ Transcription Factor Staining Buffer Set, eBioscience) overnight at 4°C. After permeabilization, cells were stained for intracellular markers for 1hr at RT. Data were acquired on a BD LSR II flow cytometer (BD Biosciences). All compensation and gating analysis were performed using FlowJo 9.6.3 (TreeStar, Ashland, OR).

Venkatasubramanian S., Tripathi D., Tucker T., Paidipally P., Cheekatla S., Welch E., Raghunath A., Jeffers A., Tvinnereim A.R., Schechter M.E., Andrade B.B., Mackman N., Idell S, & Vankayalapati R. (2015). TISSUE FACTOR EXPRESSION BY MYELOID CELLS CONTRIBUTES TO PROTECTIVE IMMUNE RESPONSE AGAINST Mycobacterium tuberculosis INFECTION. European journal of immunology, 46(2), 464-479.

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Multiparametric Flow Cytometry Analysis

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The following antibodies were utilized: CD3 (UCHT1), CD4 (SK3), CD45RA (L48), CD8 (SK1), CD16 (3G8), CD33 (P67.6), pSTAT1 (pY701) (4a), total STAT1 (1/stat1), CXCR4 (12G5), IFNγR1(GIR208) (BD Biosciences, San Jose, CA, USA), CD14 (HCD14), CSF1R (94D21E4), CCR2 (K036C2), VEGFR2 (7D46), MRC1 (15-2), CD163 (GHI/61) (Biolegend, San Diego, CA, USA), LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies, Carlsbad, CA, USA). The IFNγ signaling response was expressed as the IFNγ induced median fluorescence intensity (MFI) minus the unstimulated MFI of pSTAT1. Flow cytometry was performed using Fortessa Flow Cytometers (BD Biosciences). Flow cytometry data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA.).

Wang L., Simons D.L., Lu X., Tu T.Y., Avalos C., Chang A.Y., Dirbas F.M., Yim J.H., Waisman J, & Lee P.P. (2020). Breast cancer induces systemic immune changes on cytokine signaling in peripheral blood monocytes and lymphocytes. EBioMedicine, 52, 102631.

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Pancreatic Tumor Dissociation and Flow Cytometry Analysis

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Pancreatic tumors harvested from PKT mice were enzymatically digested using the solution of 0.6 mg/mL of collagenase P (Roche), 0.8 mg/mL Collagenase V (Sigma-Aldrich), 0.6 mg/mL soybean trypsin inhibitor (Sigma-Aldrich), and 1,800 U/mL DNase I (Thermo Fisher Scientific) in RPMI medium for 20–30 minutes at 37°C. Samples were then washed and resuspended in cold PBS (supplemented with 2 mmol/L Etheylenediaminetetraacetic acid (EDTA) and 0.5% BSA), followed by straining through a 40 μm mesh filter to obtain single-cell suspension. Samples were frozen at −80°C until further use. Prior to flow cytometry staining, samples were thawed, washed, and incubated with mouse FcR blocking reagent (Miltenyi Biotec) before subsequent staining with fluorescently conjugated antibodies listed in Supplementary Table S2. Live/dead cell discrimination was performed using Live/Dead Fixable Blue Dead Cell Stain (Life Technologies), and for intracellular staining, cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences) as per the manufacturer's instructions. Flow cytometric data acquisition was performed on Cytek Aurora and analyzed using FlowJo v10 software (BD Life Sciences). Gating strategies are depicted in Supplementary Fig. S3.

Mehra S., Garrido V.T., Dosch A.R., Lamichhane P., Srinivasan S., Singh S.P., Zhou Z., De Castro Silva I., Joshi C., Ban Y., Datta J., Gilboa E., Merchant N.B, & Nagathihalli N.S. (2023). Remodeling of Stromal Immune Microenvironment by Urolithin A Improves Survival with Immune Checkpoint Blockade in Pancreatic Cancer. Cancer Research Communications, 3(7), 1224-1236.

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Multiparameter Flow Cytometry Analysis

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The following antibodies were used: V450‐conjugated CD3 (clone UCHT1), fluorescein isothiocyanate (FITC)–conjugated CD19 (HIB19), phycoerythrin (PE)–conjugated CD73 (AD2), FITC‐conjugated CD66B (G10F5) (all from BD Biosciences), allophycocyanin (APC)–conjugated CD8 (SK1), PE–Cy7–conjugated CD39 (eBioA1), Alexa 488–conjugated interferon‐γ (IFNγ) (42.B3), APC‐conjugated FoxP3 (PCH101), eFluor 450–conjugated Ki‐67 (20Raj1), PerCP–Cy5.5–conjugated CD14 (61D3), FITC‐conjugated CD14 (61D3) (all from eBioscience), Qdot 605–conjugated CD4 (S3.5) (Invitrogen), and APC‐ and perforin (dG9)/FITC–conjugated CD26 (BA5b) (Biolegend). Dead cells were excluded using Live/Dead fixable blue DEAD Cell Stain (Life Technologies). Flow cytometry data were acquired on an LSRII (BD PharMingen) and analyzed with FlowJo version 7.6.4 (Tree Star).

Botta Gordon‐Smith S., Ursu S., Eaton S., Moncrieffe H, & Wedderburn L.R. (2015). Correlation of Low CD73 Expression on Synovial Lymphocytes With Reduced Adenosine Generation and Higher Disease Severity in Juvenile Idiopathic Arthritis. Arthritis & Rheumatology (Hoboken, N.j.), 67(2), 545-554.

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Characterization of Peripheral Immune Cells

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Peripheral blood mononuclear cells were assessed for expression of T-cell, B-cell, natural killer (NK) cell, NK-T-cell and monocyte surface markers by flow cytometry [CD3, CD19, CD16, CD14, CD4, CD8, CD45RA and CD45RO, using the following fluorochromes: FITC, phycoerythrin, PC7, allophycocyanin, Peridinin chlorophyll protein-Cy5.5, Qdot605, allophycocyanin-Cy7 and V450, purchased from: BD (Franklin Lakes, NJ), Life Technologies (Carlsbad, CA), eBioscience (San Diego, CA) or Beckman Coulter (Brea, CA)]. TCRVb staining was performed using the iotest® Beta Mark Kit (Beckman Coulter) according to the manufacturer's instructions. LIVE/DEAD Fixable blue Dead Cell Stain (Life Technologies) was used to exclude dead cells. Cytometric analysis was performed using LSRII or FACScan (both BD) and flowjo (Treestar Inc., Ashland, OR).

Wu Q., Pesenacker A.M., Stansfield A., King D., Barge D., Foster H.E., Abinun M, & Wedderburn L.R. (2014). Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation. Immunology, 142(2), 227-236.

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Characterization of Human Nasal Dendritic Cells

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