Near ir dead cell stain

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The Near IR Dead cell stain is a fluorescent dye that selectively labels dead cells in a population. It emits in the near-infrared region of the spectrum, enabling detection of dead cells using flow cytometry or fluorescence microscopy.

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Lab products found in correlation

Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (4 mentions) Anti granzymeb alexafluor700, by BD (2 mentions) Anti perforin pacific blue, by BioLegend (2 mentions) Anti perforin fitc, by BD (2 mentions) Anti granzyme b apc, by Thermo Fisher Scientific (2 mentions) Macsquant, by Miltenyi Biotec (2 mentions) Anti cd16 32 2.4g2, by BD (2 mentions) Fortessa, by BD (2 mentions) Phosflow perm buffer 3, by BD (2 mentions) Calcein blue, by Thermo Fisher Scientific (1 mentions) Tryple, by Thermo Fisher Scientific (1 mentions) Stain buffer, by BD (1 mentions) Ultracomp ebeads compensation beads, by Thermo Fisher Scientific (1 mentions) Moflo astrios flow cytometer, by Beckman Coulter (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Fc block, by BD (1 mentions) Anti ki67 fitc, by BD (1 mentions) Anti cd4 viogreen, by Miltenyi Biotec (1 mentions) Anti granzyme a percp cy5, by BioLegend (1 mentions) Anti cd69 fitc, by Thermo Fisher Scientific (1 mentions)

11 protocols using near ir dead cell stain

Mammary Gland Flow Cytometry

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For flow cytometry experiments, mammary glands were excised and digested as described for organoid experiments. Following digestion at 37 °C, samples were dissociated into single cells using TrypLE (Gibco, 12604-021), quenched with DMEM/10% FBS, and washed with PBS. Cells were then put through a 40 µM (Fisher, 22-363-547) or 70 µM strainer (Corning, 352350). For mammary gland profiling, live/dead staining was performed using a near-IR dead cell stain (Invitrogen, L34976), followed by blocking using Fc block (BD Biosciences, 553142) in BD stain buffer (BD, 554656). Samples were then incubated in primary antibodies at the specified concentrations for 30 min at 4 °C, washed twice, and resuspended in PBS. Compensation was performed using UltraComp eBeads Compensation Beads (Invitrogen). Flow cytometry was performed using a BD LSRII flow cytometer using FACS Diva 6.2.1 (BD Biosciences) and analysis was performed using FlowJo v10.7.1 (BD Biosciences). All antibodies used for flow cytometry are outlined in Supplementary Table 2. For FACS experiments, live/dead staining was performed using Calcein blue (Invitrogen, C1429) at a concentration of 1 µM. Cell sorting was performed on a Moflo Astrios flow cytometer (Beckman Coulter).

Kern J.G., Tilston-Lunel A.M., Federico A., Ning B., Mueller A., Peppler G.B., Stampouloglou E., Cheng N., Johnson R.L., Lenburg M.E., Beane J.E., Monti S, & Varelas X. (2022). Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas. Nature Communications, 13, 7198.

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Multiparametric Flow Cytometry of Immune Cells

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For immunofluorescence staining, dead cells were excluded with the Near-IR Dead-Cell stain (Invitrogen). Antibodies used were: anti-CD3 PE-Cy7 or APC, anti-CD8 PerCP-Cy5.5 or eFluor 450, anti-CD69 FITC (eBioscience); anti-CD161 PE or APC, anti-CD4 VioGreen (Miltenyi Biotec); anti-Vα7.2 PE or FITC or PE-Cy7, anti-CD107α PE-Cy7, anti-Granzyme A PerCP-Cy5.5, anti-Perforin Pacific Blue, anti-granulysin PE, anti-FasL PE (BioLegend); anti-Granzyme B AlexaFluor700, anti-Perforin FITC, anti-Ki67 FITC (BD Biosciences), anti-Granzyme B APC (Invitrogen); anti-Granzyme K FITC (Immunotools); anti-T-bet PE (Santa Cruz Biotechnology); anti-Granzyme A FITC, anti-Blimp1 AlexaFluor488 (R&D); anti-CD8β PE (Beckman Coulter).
Data were collected on the flow cytometers LSRII (BD Biosciences) or MACSQuant (Miltenyi Biotec), and was analyzed using FlowJo v9.6 (TreeStar). For ImageStream analysis, see Supplementary Methods.

Kurioka A., Ussher J.E., Cosgrove C., Clough C., Fergusson J.R., Smith K., Kang Y.H., Walker L.J., Hansen T.H., Willberg C.B, & Klenerman P. (2014). MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets. Mucosal Immunology, 8(2), 429-440.

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Quantitative Analysis of Immune Cell Markers

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Skin cells were incubated with 50 µg/ml human IgG (Sigma), Near IR Dead cell stain (Invitrogen), and fluorescent antibodies (Supporting Information: Table S2) before fixation with Foxp3/Transcription Factor Buffer Staining Set (eBioscience), and analysis using a BD Fortessa flow cytometer and FlowJo (TreeStar).

Linley H., Jaigirdar S., Mohamed K., Griffiths C.E, & Saunders A. (2022). Reduced cutaneous CD200:CD200R1 signaling in psoriasis enhances neutrophil recruitment to skin. Immunity, Inflammation and Disease, 10(7), e648.

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Flow Cytometric Analysis of Immune Cells

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Dead cells were excluded with the Near‐IR Dead‐Cell stain (Invitrogen, Paisley, UK). Antibodies used were: anti‐CD3 phycoerythrin‐cyanin7 (PE‐Cy7) or allophycocyanin (APC), anti‐CD8 peridinin chlorophyll (PerCP)‐Cy5.5 or eFluor 450 (eBioscience, Hatfield, UK); anti‐CD161 PE or APC, anti‐CD8 VioGreen, anti‐interferon (IFN) fluorescein isothiocyanate (FITC) (Miltenyi Biotec, Surrey, UK); anti‐V7.2 PE or FITC or PECy7, anti‐perforin Pacific Blue, anti‐CD243/MDR1 PE (Biolegend, London, UK); anti‐granzyme B AlexaFluor700, anti‐perforin FITC, anti‐IFN AlexaFluor700 (BD Biosciences, Oxford, UK) and anti‐granzyme B APC (Invitrogen). For intracellular antibody staining cells were stained with the forehead box protein 3 (FoxP3)/transcription factor staining buffer set (eBioscience, Birmingham, UK).
Data were acquired on a MACSQuant (Miltenyi Biotec) or LSRII (BD Bioscience) and analysed using FlowJo software version 9 (Treestar, Inc., Ashland, OR, USA).

Fergusson J.R., Ussher J.E., Kurioka A., Klenerman P, & Walker L.J. (2018). High MDR‐1 expression by MAIT cells confers resistance to cytotoxic but not immunosuppressive MDR‐1 substrates. Clinical and Experimental Immunology, 194(2), 180-191.

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Multiparametric Flow Cytometry Analysis

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Following the initial target cell count, leftover samples from each condition were combined for a surface staining of degranulation and activation. Cells were transferred to 96 u-bottom plate (Sarstedt), washed with PBS, and stained with fixable Near-IR Dead Cell Stain (Invitrogen) as well as αCD56-BUV395 (BD Bioscience, clone: NCAM16.2, dilution 1:100), αCD16-BV711 (BioLegend, clone: 3G8, dilution 1:100), αCD107a-BV421(BioLegend, clone: H4A3, dilution 1:100), αCD69-PE (BioLegend, clone: FN50, dilution 1:100) in PBS supplemented with 2% FBS for 30 min at 4 °C. Cells were washed twice prior to intracellular staining. Perforin staining was performed using BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) and αPerforin-APC (BioLegend clone:dG9, dilution 1:50) according to the manufacturer’s protocol. Cells were fixed with 1×CellFIX (BD Biosciences) and stored at 4 °C in PBS until flow cytometry.

Aziz U.B., Saoud A., Bermudez M., Mieth M., Atef A., Rudolf T., Arkona C., Trenkner T., Böttcher C., Ludwig K., Hoelzemer A., Hocke A.C., Wolber G, & Rademann J. (2024). Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins. Nature Communications, 15, 3537.

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Multiparametric Immune Cell Analysis

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Cells were incubated with 0.5 µg/ml anti‐CD16/32 (2.4G2, BD Bioscience), Near IR Dead cell stain (Invitrogen) and fluorescently labeled antibodies (see Supporting Information: Table S4). For cytokine analysis, cells were cultured for 4 h with 10 μM Brefeldin A before staining. Cells were fixed and permeabilized with Foxp3/Transcription Factor Buffer Staining Set (eBioscience). For apoptosis the Annexin V Apoptosis Detection Kit eFluor 450 (Invitrogen) was used. ROS was assayed using Total ROS Assay Kit 520 nm (Invitrogen). For pDok cells were fixed with BD PhosFlow Lyse/Fix buffer then Foxp3/Transcription Factor Buffer Staining Set (eBioscience) before a permeabilization with BD PhosFlow Perm Buffer III and staining with pDok1 Y398, then anti‐rabbit AF488. Lymph node cells were stained rather than skin to avoid potential effects of lengthy enzymatic digestion of skin on pDok1 levels. Cells were analyzed using a BD Fortessa or LSRII flow cytometer and FlowJo (TreeStar).

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Comprehensive Flow Cytometry Analysis of Immune Cells

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Cells were incubated with 0.5 µg/ml anti-CD16/32 (2.4G2, BD Bioscience) and Near IR Dead cell stain (Invitrogen) prior to staining with fluorescently labelled antibodies. Cells were fixed with Foxp3/Transcription Factor Buffer Staining Set (eBioscience) for between 30 min and 16 hr at 4°C.
For cytokine analysis, 10 µM Brefeldin A was added to cell cultures for 4 hr prior to staining for cell surface markers as described above. After overnight fixation, cells were permeabilized with Foxp3/Transcription Factor Buffer Staining Set (eBioscience) and were stained with intracellular markers or cytokines. Cells were analysed on a BD Fortessa or LSRII flow cytometer. Data were analysed using FlowJo (TreeStar). Antibodies are detailed in Table S1.
For pSTAT3 staining, inguinal, axillary and brachial LN cells were stained for surface markers, then stimulated with 100 ng/ml IL-23 (Biolegend) for 15 min. Cells were fixed with Phosflow Fix buffer I (BD Biosciences) at 37 °C for 10 min, before permeabilisation at 4 °C in Phosflow Perm Buffer III (BD Biosciences) for 30 min, staining with PE conjugated pSTAT3 (pY705) (BD Bioscience clone 4/P-STAT3) at room temperature for 30 min. Data were normalized to the mean stimulated WT data. ILCs were gated as live, single cell, CD45⁺, Lin^- (Ter119, F4/80, CD11b, CD11c, FceRIa, Gr1, CD19), CD3^-, TCRb^-, TCRgd^-, CD90.2⁺ CD127⁺.

Linley H., Ogden A., Jaigirdar S., Buckingham L., Cox J., Priestley M, & Saunders A. (2023). CD200R1 promotes interleukin-17 production by group 3 innate lymphoid cells by enhancing signal transducer and activator of transcription 3 activation. Mucosal Immunology, 16(2), 167-179.

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Flow Cytometry Analysis of Pancreatic Islet Cells

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FACS analysis was performed essentially as described [17 (link)]. After overnight incubation, 250–300 islets of the same genotype were handpicked and dissociated into single cells by repeated pipetting in 150 μl of Hank’s based cell dissociation buffer (Invitrogen) containing 0.1 % BSA and 0.1 % trypsin. The reaction was stopped with the addition of FBS (20 μl; Seralab). Cells were incubated in near-IR dead cell stain (Life technologies) for 20 min. at 4 °C, washed with PBA (PBS, 1 % BSA, 0.1 % azide) and fixed in 2 % (w/v) PFA for 10 min. and then washed twice with PBA and once with Saponin (0.025 % in PBA) before 10 min incubation with Saponin at room temperature. Cells were incubated with primary antibodies against insulin or glucagon (see Table 2) before incubation with secondary antibodies (anti-mouse AF 405, anti-guinea pig AF 488, anti-rabbit AF 640) for 20 min and resuspension in PBA. The samples were run on a BD Fortessa Flow Cytometer (BD Bioscience).Table 2

Antibodies

Primary
Antibody	Species	Company	Dilution
ZnT8	Rabbit	Mellitech	1:200
Glucagon	Mouse	Sigma	1:1000
Glucagon	Rabbit	Santa Cruz	1:200
Insulin	Guinea pig	Santa Cruz	1:200
Secondary
Antibody	Species	Company	Dilution
Anti-mouse Alexa 568	Goat	Invitrogen	1:1000
Anti-rabbit Alexa 488	Goat	Invitrogen	1:1000
Anti-guinea pig Alexa 488	Donkey	Invitrogen	1:1000
Anti-mouse Alexa 488	Donkey	Invitrogen	1:1000
Anti-rabbit 568	Goat	Invitrogen	1:1000

Solomou A., Philippe E., Chabosseau P., Migrenne-Li S., Gaitan J., Lang J., Magnan C, & Rutter G.A. (2016). Over-expression of Slc30a8/ZnT8 selectively in the mouse α cell impairs glucagon release and responses to hypoglycemia. Nutrition & Metabolism, 13, 46.

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Multicolor Flow Cytometry of Islet Cells

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Islets were dissociated into single cells as described previously (26 (link)), washed in PBS, and centrifuged at 600 × g for 2 min. Cells were incubated in 50 μl of near-IR dead cell stain (1:1000; Life Technologies) for 20 min at 4 °C, washed with PBA (PBS, 1% BSA, 0.1% azide), and fixed in 2% paraformaldehyde for 10 min at room temperature. Cells were then washed twice with PBA and once with saponin (0.025% in PBA) before a 10-min incubation with saponin at room temperature. Cells were incubated with primary antibodies against mouse ZnT8 (Mellitech, Grenoble, France) and insulin and glucagon (DAKO and Santa Cruz Biotechnology, respectively) at 1:100 dilution in saponin for 20 min. After two further washes in saponin, cells were incubated with secondary antibodies (anti-mouse Alexa Fluor 405, anti-guinea pig Alexa Fluor 488, anti-rabbit Alexa Fluor 640) for 20 min. Two final washes in saponin were performed before resuspension in PBA. The samples were processed on a BD LSRFortessa flow cytometer (BD Biosciences).

Solomou A., Meur G., Bellomo E., Hodson D.J., Tomas A., Migrenne Li S., Philippe E., Herrera P.L., Magnan C, & Rutter G.A. (2015). The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion. The Journal of Biological Chemistry, 290(35), 21432-21442.

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Detection of AH1-Specific CD8+ T Cells

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For detection of AH1 positive CD8⁺ T cells, tumor and lymph nodes were excised 24 h after treatment of mice as described above. Tumor samples were digested for 2 h at 37 °C with 1 mg/mL collagenase II and 0.1 mg/mL DNase I in RPMI medium supplemented with 1 % Antibiotic-Antimycotic. The filtered tumor cell suspension was diluted to 5 x 10⁶ cells per mL. Lymph nodes were collected in sterile PBS and mechanically opened. The cell suspension was washed with Red Blood Cell Lysis buffer and isolated lymphocytes were diluted to 1 x 10⁶ cells per mL. 100 μL of tumor cell or lymphocyte suspension were stained for 1 hour at 4 °C with a PE labelled antibody against CD90.2 (53-2.1, Biolegend), a FITC labelled antibody against CD8⁺ T cells (53-6.7, Biolegend), a APC labelled H-2L^d tetramer loaded with AH1 peptide(28 (link)) and a live/dead fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific). Following fixation and permeabilization, cells were analyzed on a CytoFLEX cytometer (Beckmann Coulter) and data were processed using FlowJo (v.10, Tree Star).

Ziffels B., Stringhini M., Probst P., Fugmann T., Sturm T, & Neri D. (2019). Antibody-based delivery of cytokine payloads to carbonic anhydrase IX leads to cancer cures in immunocompetent tumor-bearing mice. Molecular cancer therapeutics, 18(9), 1544-1554.

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