Live dead stain

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The LIVE/DEAD stain is a fluorescent dye-based solution used to distinguish between live and dead cells in a sample. It provides a simple, reliable, and rapid method for assessing cell viability.

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Lab products found in correlation

Lsrfortessa, by BD (13 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions) Lsrii flow cytometer, by BD (9 mentions) Golgistop, by BD (6 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions) Cytofix cytoperm, by BD (6 mentions) Lsr 2, by BD (5 mentions) Ionomycin, by Merck Group (5 mentions) Facsdiva software, by BD (5 mentions) Golgiplug, by BD (5 mentions) Kaluza software, by Beckman Coulter (4 mentions) Facsaria, by BD (4 mentions) Facscanto 2, by BD (4 mentions) Facsaria 2, by BD (4 mentions) Lsrfortessa cell analyzer, by BD (3 mentions) Fortessa, by BD (3 mentions) Facscalibur, by BD (3 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions) B27 supplement, by Thermo Fisher Scientific (3 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)

164 protocols using live dead stain

Multiparameter Flow Cytometry Analysis

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Anti-mouse CD18 (LFA-1), CD326 (EpCAM), MHC-I (H-2K^b), 5E6 (anti-Ly49C/I), 4D11 (anti-Ly49G), CD8 (CD8β), CD4, CD3, TCRβ, NKp46 (CD335), NKG2D (CD314), NKG2A (16a11), NKG2A/C/E (20d5), CD27, CD11b, CD107a (1D4B), IFN-γ (XMG1.2), and Live/Dead stain were purchased from eBioscience (eBioscience, San Diego, CA, USA). Anti-NKG2D (CD314) was purchased from BioLegend (BioLegend, San Diego, CA, USA). Anti-mouse TCRβ chain was purchased from BD Biosciences (BD Biosciences, Mississauga, Ontario, Canada). PK136 (anti-NK1.1), 5E6 (anti-Ly49C/I^B6), and XMG1.2 (anti-IFN-γ) hybridomas were kind gifts from Drs. James Carlyle (Sunnybrook Research Institute, Toronto, ON), Charles Sentman (Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire), and Subash Sad (University of Ottawa, Ottawa, ON), respectively. Cell fluorescence data was acquired with a CyAN-ADP flow cytometer (Beckman Coulter) and analyzed with Kaluza software (Beckman Coulter, New Jersey, USA).
The levels of cytokines and chemokines in lung tissue homogenates were measured by bead array flow cytometry using the mouse Th1/Th2/Th17/Th22-13plex FlowCytomix multiplex kit and mouse chemokine 6plex kit (eBioscience, San Diego, CA, USA).

Mahmoud A.B., Tu M.M., Wight A., Zein H.S., Rahim M.M., Lee S.H., Sekhon H.S., Brown E.G, & Makrigiannis A.P. (2016). Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion. PLoS Pathogens, 12(2), e1005446.

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Lung Cell Isolation and Intracellular Cytokine Analysis

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After lavage, lungs were removed and the upper right lobe was minced and digested in 2 ml RMPI + 0.5 mg/ml Liberase DL (Roche Diagnostics) and 0.5 mg/ml DNAse I (Sigma) at 37ºC for 30 min. Cells were then passed over a 70 μm cell strainer. Cells were washed with PBS and counted. ~1×10⁶ cells were stimulated with 50 ng/mL PMA + 500 ng/mL ionomycin for 6h. After 2 hours, Brefeldin A was added. CD4⁺ T cells were stained with a viability dye (eBioscience Live/Dead stain) and antibodies for CD3, CD4, CD44, and γδTCR; intracellular staining for IL-13, IL-17A, and IFNγ was performed after fixation and permeabilization according to manufacturer’s instructions (eBioscience). Foxp3 staining was performed on separate cells that had not been restimulated ex vivo.

Patterson A.R., Bolcas P., Lampe K., Cantrell R., Ruff B., Lewkowich I., Hogan S.P., Janssen E.M., Bleesing J., Khurana Hershey G.K, & Hoebe K. (2018). Loss of Gimap5 promotes pathogenic CD4+ T cell development and allergic airway disease: Gimap5-deficiency predisposes CD4+ T cells to Th17 polarization. The Journal of allergy and clinical immunology, 143(1), 245-257.e6.

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Treg, Th1, and Th17 Cell Analysis by Flow Cytometry

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To determine Treg frequencies, single cell suspensions prepared as above were blocked with rat anti-mouse CD16/CD32 Fc block (BD Pharmingen, San Jose, CA, USA), stained with cell surface antibodies, then fixed and permeabilized with the FoxP3 staining buffer kit (eBioscience, San Diego, CA, USA) before staining with any intracellularly directed antibodies. The following anti-mouse antibody panel was used: CD25-PE, Helios-efluor450, Foxp3-APC, CD4-FITC, and Live/Dead stain conjugated to efluor780 (all eBioScience). For Th1 and Th17 cell detection, single cell suspensions were stimulated overnight (16 hours) with 500 ng/mL phorbal myristate acetate (PMA) and 2 μg/mL ionomycin (I) in culture media (10% FBS, RPMI, penicillin, streptomycin, β-mercaptoethanol) at 37°C in the presence of GolgiStop (BD Biosciences, San Jose, CA, USA) for the last 4 hours. Cells were then fixed and permeabilized using an intracellular cytokine detection kit (BD Biosciences). They were stained with the following anti-mouse antibody panel: CD4-PE-Cy7, interferon-γ (IFN-γ)-FITC (BD Biosciences), TNF-α-PE (eBioscience), IL-17A-AlexaFluor647 (BD Pharmingen), and Live/Dead stain as above. Flow cytometric data were acquired on a LSRFortessa cell analyzer (BD Biosciences).

Nakamura Y.K., Metea C., Karstens L., Asquith M., Gruner H., Moscibrocki C., Lee I., Brislawn C.J., Jansson J.K., Rosenbaum J.T, & Lin P. (2016). Gut Microbial Alterations Associated With Protection From Autoimmune Uveitis. Investigative Ophthalmology & Visual Science, 57(8), 3747-3758.

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Identification and Quantification of Muscle Macrophages

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Single-cell suspensions from muscle were prepared by collagenase II (Worthington Biochemical Corp, Lakewood, NJ, USA) digestion followed by staining with fluorochrome-conjugated antibodies.
Antibodies used: (1) Gr1 Clone RB6-8C5 (eBioscience, ThermoFisher, Waltham, MA, USA, Cat#48-5931-82); (2) F4/80 Clone BM8 (Invitrogen, Cat# 11-4801-82); (3) CD11b Clone M1/70 (Invitrogen, ThermoFisher, Waltham, MA, USA, Cat#17-0112-81); (4) Siglec-F Clone E50-2440 (RUO), (BD BioSciences, ThermoFisher, Waltham, MA, USA, Cat# 552126); (5) CD206 Clone C068C2 (BioLegend, San Diego, CA, USA, Cat#141701) and live dead stain (eBioscience). Data acquisition was performed on a FACS Aria or Fortessa (BD Biosciences). UltraComp eBeads (Cat#01333342, Invitrogen) were used to generate single-stain controls. Data was analyzed using FlowJo software (version 10.2) and gating strategies as previously described [39 (link)] to discriminate against dead cells, debris, and doublets were utilized. M1 macrophages were defined as Gr1^low-med, F4/80⁺, and CD11b⁺.

Lynch C.A., Acosta S.A., Anderson D.M., Rogers G.E., Wilson-Rawls J, & Rawls A. (2024). The Transcription Factor Mohawk Facilitates Skeletal Muscle Repair via Modulation of the Inflammatory Environment. International Journal of Molecular Sciences, 25(9), 5019.

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Multiparametric Immunophenotyping of Mouse T-Cells

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Rat Anti-Mouse IFNγ clone XMG1.2, Invitrogen, Cat #: 12-7311-82, PE (1 μL/1 × 10⁶ cells).
Rat Anti-Mouse TNFα clone MP6-XT22, eBioscience, Cat#17-7321-82, APC (1.25 μL/1 × 10⁶ cells).
Rat Anti-Mouse IL2 clone JES6-5H4, eBioscience, Cat#48-7021-82, eFluor450 (1.25 μL/1 × 10⁶ cells).
Rat Anti-Mouse IL4 clone 11B11, BioLegend, Cat#504132, PE-Dazzle594 (1.25 μL/1 × 10⁶ cells).
Rat Anti-Mouse IL10 clone JES5-16E3, eBioscience, Cat#56-7101-82, AF700 (1.25 μL/1 × 10⁶ cells).
Rat Anti-Mouse CD4 clone GK1.5, BioLegend, Cat#100451, BV605 (2.5 μL/1 × 10⁶ cells).
Rat Anti-Mouse CD8 clone 53-6.7, BD, Cat#563332, BV786 (0.75 μL/1 × 10⁶ cells).
Live/dead stain, eBioscience Cat#65-0866-18, eFluor506 (1:1000).

Rappaport A.R., Hong S.J., Scallan C.D., Gitlin L., Akoopie A., Boucher G.R., Egorova M., Espinosa J.A., Fidanza M., Kachura M.A., Shen A., Sivko G., Van Abbema A., Veres R.L, & Jooss K. (2022). Low-dose self-amplifying mRNA COVID-19 vaccine drives strong protective immunity in non-human primates against SARS-CoV-2 infection. Nature Communications, 13, 3289.

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Multiparameter Flow Cytometric Profiling

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Red blood cells were lysed in ammonium chloride buffer and centrifuged for 10 min at 2500 RPM. Cell pellets were re-suspended and viable cells were counted using Trypan blue staining solution on a haemocytometer (Nikon, Tokyo Japan). Flow cytometry was performed using a method adapted from Barnett-Vanes et al., (2015) . Briefly, cells were stained with live-dead stain (eBioscience), blocked with anti-cd32 blocking antibody and stained with antibodies CD43 PE (Biolegend) and His48 FITC (Biolegend) for neutrophils and monocytes, CD161 APC (Biolegend) for NK Cells and CD3 VioGreen (Miltenyi Biotec) for T Cells, in buffer containing PBS, BSA and Azide [29] . Cells were fixed in BD Cell Fix and analysed using a multi-parameter flow cytometer (Fortessa LSR BD Biosciences New Jersey USA). Flow cytometric compensation was performed using fluorescent compensation beads (OneComp eBeads, eBioscience San Diego USA).

Eftaxiopoulou T., Barnett-Vanes A., Arora H., Macdonald W., Nguyen T.T., Itadani M., Sharrock A.E., Britzman D., Proud W.G., Bull A.M, & Rankin S.M. (2016). Prolonged but not short-duration blast waves elicit acute inflammation in a rodent model of primary blast limb trauma. Injury, 47(3).

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Comprehensive Lymphocyte Phenotyping

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Lymphocytes from PB were isolated by density gradient centrifugation (Lymphocyte Separation Medium, Cellgro, MA) (18 (link), 19 (link)). T and B-lymphocytes were quantified for their regulatory function (CD25), activation (CD38+, CD69+, HLA-DR+), proliferation (Ki67+), trafficking markers (CD62L+), and naive/memory distribution (CD28+ and CD95+). For cell surface staining, 100 μl of EDTA blood was stained with directly conjugated monoclonal antibodies (MAbs) using a whole blood lysis staining protocol (19 (link)–21 (link)). Cells were first stained with live/dead stain (Invitrogen). MAbs used for this study were anti-CD3, anti-CD4, anti-CD8, anti-CD62L, anti-CD95, anti-HLA-DR, anti-CD25 and anti-CD69, anti-CD38, anti-CD20, and anti-Ki67 antibodies (Supplemental Table S1). For detecting Ki67+ proliferating cells, the intracellular staining protocol was performed as reported earlier (22 (link), 23 (link)). After staining cells were fixed in BD Stabilizing and fixative buffer and at least 30,000 events were collected from each sample by lymphocyte gating and the data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Pahar B., Baker K.C., Jay A.N., Russell-Lodrigue K.E., Srivastav S.K., Aye P.P., Blanchard J.L, & Bohm R.P. (2020). Effects of Social Housing Changes on Immunity and Vaccine-Specific Immune Responses in Adolescent Male Rhesus Macaques. Frontiers in Immunology, 11, 565746.

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Multicolour Flow Cytometry Analysis

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Cells were blocked in anti-CD16/CD32 (eBioscience) prior to staining with anti-CD11b-V450, CD19-PE or Gr1-PE, CD3-PE-Cy™7, CD45-PerCP-Cy™5.5 or CD19-PerCP-Cy™5.5, Ly-6C-APC (all from BD) and F4/80-FITC (eBioscience, Hatfield, UK). Lamina propria cells were incubated with the LIVE/DEAD^® stain (Invitrogen) prior to staining above. Cells were run on a BD LSRFortessa or LSR II after optimisation with compensation particles (BD) and analysed using FlowJo (Tree Star, Ashland, OR, USA).

Chew T.S., O'Shea N.R., Sewell G.W., Oehlers S.H., Mulvey C.M., Crosier P.S., Godovac-Zimmermann J., Bloom S.L., Smith A.M, & Segal A.W. (2015). Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis. Disease Models & Mechanisms, 8(8), 817-829.

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Peripheral Blood Mononuclear Cell Immunophenotyping

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Surface Staining: Harvested PBMCs were stained with fixable Live/Dead stain (Invitrogen) in PBS for 30 min on ice. Cells were washed and stained with surface markers in FACS buffer (PBS/1%BSA /2mM EDTA) for 30 min on ice.
Fixation and staining of intracellular markers: For the effector panel, cells were washed prior to fixation in 4% PFA for 5 min, 37 C. Cells were washed in FACS buffer, and resuspended in permeabilization buffer (eBioscience, San Diego, CA) for 20 mins at room temperature. Antibodies against intracellular targets were added and cells were incubated for 45 mins, on ice. For the regulatory panel, cells were washed with FACS buffer, and fixed with Foxp3 Fix/Perm buffer (eBioscience, San Diego, CA) for 30 mins, on ice. After washing in permeabilization buffer, cells were stained with antibodies detecting intracellular markers in permeabilization buffer for 45 mins, on ice.
Stained and permeabilized cells were washed twice in permeabilization buffer before resuspension in FACS buffer, and subsequently acquired on an LSR Fortessa (BD Biosciences). Antibodies used for the regulatory panel are shown in Table 2.

Agashe C., Chiang D., Grishin A., Masilamani M., Jones S.M., Wood R.A., Sicherer S.H., Burks A.W., Leung D.Y., Dawson P., Sampson H.A, & Berin M.C. (2017). Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells. Journal of immunological methods, 449, 23-27.

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Multiparametric Flow Cytometry Analysis

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Tumor and blood were stained without stimulation for the following surface markers: CD3, PD-1, CD45RO, CD8, CD62L, CD28 (BD), and for CD19 and CD4(eBioscience). Cytokine production was stimulated with phorbol 12-myristate 12-acetate(PMA, 50 ng/mL) and ionomycin(1 mM) in the presence of a Golgi blocker(Brefeldin A, 1 mL/mL) for 4–12 hours for intracellular cytokine analysis. All samples were labeled with live/dead stain (Invitrogen) prior to antibody labeling. To detect IL-17 producing T cells and Treg, we labeled the cells before fixing and permeabilizing(BioLegend FOXP3 fix/perm buffer set) with CD3, CD4, CD127(eBiosciences), CD8, and CD25(BD), and then staining for IL-17(eBioscience) and FOXP3(BioLegend).
IFN-γ production was analyzed using a separate panel by labeling the cells with CD45, CD3, CD16, CD19(BD), and CD56(BioLegend), before fixing and permeabilizing the cells with the eBioscience fix/perm buffer set and then staining with IFN-γ (eBioscience). Flow cytometry for all samples and controls was conducted using an LSRII flow cytometer(BD). Data analysis and compensation using single-color controls were performed using FlowJo(Tree Star, Ashland, OR).

Shibuya K.C., Goel V.K., Xiong W., Sham J.G., Pollack S.M., Leahy A.M., Whiting S.H., Yeh M.M., Yee C., Riddell S.R, & Pillarisetty V.G. (2014). Pancreatic Ductal Adenocarcinoma Contains an Effector and Regulatory Immune Cell Infiltrate that Is Altered by Multimodal Neoadjuvant Treatment. PLoS ONE, 9(5), e96565.

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