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Live dead fixable cell stain

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The LIVE/DEAD Fixable Cell Stain is a fluorescent dye that can be used to distinguish between live and dead cells in a sample. It binds to cellular proteins, emitting a fluorescent signal that can be detected using flow cytometry or microscopy techniques.

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Lab products found in correlation

Lsr 2, by BD (2 mentions) Facsymphony, by BD (2 mentions) Foxp3 staining kit, by BD (1 mentions) Cytofix cytoperm, by BD (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Transcription factor staining buffer kit, by Thermo Fisher Scientific (1 mentions) Facscanto 2 system, by BD (1 mentions) Anti cd45 apccy7, by Thermo Fisher Scientific (1 mentions) Facs lsr fortessa flow cytometer, by BD (1 mentions) Anti ly6g pe, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde 4, by Merck Group (1 mentions) Anti ly6c efluor 450, by Thermo Fisher Scientific (1 mentions) Anti cd11b pe cy7, by Thermo Fisher Scientific (1 mentions) Prism v8, by GraphPad (1 mentions)

Close spelling variants of this product

Live/Dead Fixable Blue Dead Cells Stain Kit LIVE/DEAD Fixable AQUA Dead Cells Stain Live/dead ™ Fixable Violet Dead Cells Stain Kit Fixable live/dead stain Fixable live/dead LIVE/DEAD stain

9 protocols using live dead fixable cell stain

1

Murine FOXP3+ T-cell Phenotyping

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Single-cell suspensions were stained as described: intracellular CTLA-4 and intranuclear transcription factor staining for murine FOXP3 was carried out using the FOXP3 staining kit (BD Biosciences) following the manufacturer's recommendations. Dead cells were excluded from all analyses using fixable LIVE/DEAD cell stain (Thermo Fisher Scientific). Data were acquired on a BD LSRII and analyzed using FlowJo software v10.7.1. Antibodies used for phenotyping are listed in Table S2. Experiment was repeated three times with different donor cells for CAR transductions for each repeat.
Arjomandnejad M., Sylvia K., Blackwood M., Nixon T., Tang Q., Muhuri M., Gruntman A.M., Gao G., Flotte T.R, & Keeler A.M. (2021). Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells. Molecular Therapy. Methods & Clinical Development, 23, 490-506.
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2

Ex Vivo Cytokine Analysis of Leukocytes

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Cells were harvested from the ear and stained with fixable live/dead cell stain (ThermoFisher) (42 (link)). For ex vivo intracellular staining of cytokines, brefeldin A (1 μg/ml) was added to all media and wash buffers to block cytokine secretion (43 (link)). Leukocytes were stained with Abs (Biolegend) to CD45 (30-F11), CD4 (RM4-5) and Thy1.1 (H1S51), fixed with cytofix/cytoperm (BD Biosciences) and stained with anti-IFNγ Ab (XMG1.2). Analyzed by FACS using the BD LSR II.
Fernandes N.R., Reilly N.S., Schrock D.C., Hocking D.C., Oakes P.W, & Fowell D.J. (2020). CD4+ T Cell Interstitial Migration Controlled by Fibronectin in the Inflamed Skin. Frontiers in Immunology, 11, 1501.
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3

Measuring NK Cell Cytotoxicity against YAC-1 Targets

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YAC-1 NK sensitive target cells (32 (link)) were labelled with CFSE (Invitrogen) as previously described (30 ). To 10,000 labelled target cells spleen cells were added at different attacker:target ratios in round bottom 96 well plates and the plates were centrifuged for 3 minutes at 1500 rpm before incubation for 16 at 37°C hours. The plates were centrifuged again and the supernatant removed, the cells resuspended by vortexing and 50μl fixable live/dead cell stain added (Molecular Probes). After 20 mins at 4°C 100μl FACs buffer was added to each well, the plate was centrifuged, the supernatant removed, cells resuspended and 200μl of FACs buffer added. An aliquot of 100μl was removed from each well and added to an equal volume of medium containing a known number (~104) of fluorescent microbeads. The ratio of viable target cells to beads was determined by flow cytometry and % cytotoxicity calculated by the formula:
%cytotoxicity=1−(ratio of viable target cells∕beads at each A:T ratio)ratio of viable target cells alone∕beads×100
Beverley P.C., Ruzsics Z., Hey A., Hutchings C., Boos S., Bolinger B., Marchi E., O'Hara G., Klenerman P., Koszinowski U.H, & Tchilian E.Z. (2014). A Novel Murine Cytomegalovirus Vaccine Vector Protects against Mycobacterium tuberculosis. The Journal of Immunology Author Choice, 193(5), 2306-2316.
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4

Flow Cytometric Analysis of Rhesus Macaque and Human Blood

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Rhesus macaque and human blood samples were collected in EDTA tubes, and WB was used for flow cytometry. Briefly, antibody cocktails were directly added to 200 μl of WB and stained at 37°C for 20 min, after which samples were washed twice in 4 ml 1% FCS/PBS. Fixable live/dead cell stain (Molecular Probes; Invitrogen) was used according to the manufacturer’s protocol. Pellets were then fixed and permeabilized with the eBioscience Foxp3 Transcription Factor Staining Buffer Kit (Life Technologies) for at least 1 h followed by intracellular antigen staining for 30 min at 4°C. Cells were washed and samples acquired on a FACSymphony (BD Biosciences) at NIH or an LSRFortessa (BD Biosciences) at SPHCC. FACS data were analyzed using FlowJo 10 (FlowJo, LLC).
Bohrer A.C., Castro E., Hu Z., Queiroz A.T., Tocheny C.E., Assmann M., Sakai S., Nelson C., Baker P.J., Ma H., Wang L., Zilu W., du Bruyn E., Riou C., Kauffman K.D., Moore I.N., Del Nonno F., Petrone L., Goletti D., Martineau A.R., Lowe D.M., Cronan M.R., Wilkinson R.J., Barry CE I.I.I., Via L.E., Barber D.L., Klion A.D., Andrade B.B., Song Y., Wong K.W, & Mayer-Barber K.D. (2021). Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice. The Journal of Experimental Medicine, 218(10), e20210469.
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5

Comprehensive Evaluation of Rhesus Macaque Lung Granulomas

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Rhesus macaque BAL samples were passed through a 100-μm cell strainer, pelleted, and counted for analysis. Granulomas were individually resected from the lungs, and samples used for flow cytometry analysis were pushed through a 100-μm cell strainer. Aliquots from all samples were serially diluted and plated on 7H11 agar plates for CFU quantification. Alternatively, resected granulomas were fixed in 4% paraformaldehyde for later paraffin embedding. Samples were stained with surface antibody cocktails for 20 min at 4°C followed by fixable live/dead cell stain (Molecular Probes; Invitrogen). Cells were then fixed and permeabilized (eBioscience Transcription Factor Staining Buffer Kit) for at least 1 h followed by intracellular antigen staining for 30 min at 4°C. Cells were washed and samples acquired on a FACSymphony (BD Biosciences) at NIH. FACS data were analyzed using FlowJo 10 (FlowJo, LLC).
Bohrer A.C., Castro E., Hu Z., Queiroz A.T., Tocheny C.E., Assmann M., Sakai S., Nelson C., Baker P.J., Ma H., Wang L., Zilu W., du Bruyn E., Riou C., Kauffman K.D., Moore I.N., Del Nonno F., Petrone L., Goletti D., Martineau A.R., Lowe D.M., Cronan M.R., Wilkinson R.J., Barry CE I.I.I., Via L.E., Barber D.L., Klion A.D., Andrade B.B., Song Y., Wong K.W, & Mayer-Barber K.D. (2021). Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice. The Journal of Experimental Medicine, 218(10), e20210469.
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6

Tuft Cell Profiling in Intestinal Epithelium

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For analysis of tuft cells in intestinal epithelium and organoids, cells were surface stained with LIVE/DEAD Fixable Cell stain (ThermoFisher, L34957), and fluorescent conjugated antibodies against EpCAM, CD24, CD45, Siglec-F, and CD90.1 (see Supplementary Table 1 for detailed information, 1:400 in PBS) for 30 minutes. For intracellular staining of CDC42, cells were fixed/permeabilized (ThermoFisher Cat: 00-5521-00), incubated with the anti-CDC42 antibody (ThermoFisher, 1:50) for 1h in room temperature, followed by the anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, 1:400) for 1h in 4°C. IECs were defined as live CD4−CD8−Epcam+CD45low. Successfully transduced organoid cells were defined as CD90.1+. Proportion (%) of IECs in the tuft cell lineage were defined as Siglec-F+CD24+ as previously described51 (link). Flow cytometry data was analyzed with FlowJo (version 10.8.1).
Long T., Abbasi N., Hernandez J.E., Li Y., Sayed I.M., Ma S., Iemolo A., Yee B.A., Yeo G.W., Telese F., Ghosh P., Das S, & Huang W.J. (2021). RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine. Gut, 71(9), 1790-1802.
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7

Intestinal Epithelial Cell Immunophenotyping

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Intestinal epithelial cells were surface stained with LIVE/DEAD Fixable Cell stain (ThermoFisher, L34957), and fluorescent conjugated antibodies against EpCAM, TGFBR1, and IL27RA (see Supplementary Table S4 for detailed information, 1:400 in PBS) for 30 min. For intracellular staining of Ki67, cells were first fixed/permeabilized (ThermoFisher Cat: 00-5521-00) and then incubated with the anti-Ki67 antibody for 1 h at room temperature. Intestine epithelial cells were defined as live Epcam+. Flow cytometry data was analyzed with FlowJo (version 10.8.1).
Long T., Hernandez J.E., Ma S., Steele S., Luo C., Li Y., Xie Q., Telese F., Zhou B, & Huang W.J. (2023). The long non-coding RNA MALAT1 regulates intestine host-microbe interactions and polyposis. Frontiers in Cell and Developmental Biology, 11, 1168693.
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8

Single-cell Immunophenotyping by Flow Cytometry

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Adherent cells were dissociated into a single‐cell suspension using TrypLE Express Enzyme (1×), no phenol red, and subsequently treated for 30 minutes with LIVE/DEAD Fixable Cell Stain (Thermo Fisher Scientific) and then fixed using 4% wt/vol paraformaladehyde (PFA) for 10 minutes. Cells were incubated with fluorophore‐conjugated antibodies for 30 minutes in the dark, and then washed twice with phosphate buffered solution (PBS). Immunophenotyping was carried out using the BD FACSCanto II system (Becton Dickinson, Franklin Lakes, NJ) and analyzed using FlowJo software (Becton Dickinson).
Blackford S.J., Ng S.S., Segal J.M., King A.J., Austin A.L., Kent D., Moore J., Sheldon M., Ilic D., Dhawan A., Mitry R.R, & Rashid S.T. (2018). Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell‐Derived Hepatocytes for Cell‐Based Therapy. Stem Cells Translational Medicine, 8(2), 124-137.
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9

Detailed Flow Cytometry Analysis of Immune Cells

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Splenocytes were obtained from uninfected and infected animals and after RBC lysis, cells were washed in PBS, incubated with Live/Dead Fixable cell stain (ThermoFisher) for cell death exclusion, and then, with the following monoclonal antibodies: anti-CD45-APC-Cy7, anti-CD11-b-PE-Cy7, anti-Ly6C-eFluor450 and anti-Ly6G-PE (eBioscience). Samples were washed and fixed with paraformaldehyde 4% (Sigma-Aldrich) and data were acquired on a FACS LSR Fortessa flow cytometer (Becton Dickinson). Data were analyzed with FlowJo V10.0.2 (Tree Star). A forward scatter area (FSC-A) versus forward scatter height (FSC-H) gate was used to remove doublets. Viable leukocytes were selected using a Live/Dead versus CD45 gate. Expression of Ly6C and Ly6G was used to define neutrophils (Ly6CintLy6Ghigh) and monocytes (Ly6ChighLy6Gint) within CD11b+ cells. GraphPad PrismV8.0 (GraphPad-Software) was used for graphic representation.
dos Santos L.I., Torres T.A., Diniz S.Q., Gonçalves R., Caballero-Flores G., Núñez G., Gazzinelli R.T., Maloy K.J, & Ribeiro do V. Antonelli L. (2021). Disrupted Iron Metabolism and Mortality during Co-infection with Malaria and an Intestinal Gram-Negative Extracellular Pathogen. Cell Reports, 34(2), 108613.
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