Near ir dead cell stain kit

Manufactured by Thermo Fisher Scientific

The Near-IR Dead Cell Stain Kit is a fluorescent dye-based product that can be used to detect and differentiate between live and dead cells in a sample. The dye selectively binds to the DNA of dead cells, allowing their identification under near-infrared excitation and emission wavelengths.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (9 mentions) Facscanto 2, by BD (6 mentions) Fixation permeabilization kit, by BD (5 mentions) Facscanto 2 flow cytometer, by BD (4 mentions) Ionomycin, by Merck Group (4 mentions) Transcription factor buffer set, by BD (4 mentions) Phorbol myristate acetate (pma), by Merck Group (3 mentions) Brefeldin a, by Merck Group (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Quantity one colony counting software, by Bio-Rad (2 mentions) Aria facs, by Bio-Rad (2 mentions) Versadoc photodocumentation system, by Bio-Rad (2 mentions) Flow cytometry staining buffer, by BD (2 mentions) Zombie red fixable viability kit, by BioLegend (2 mentions) 7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (2 mentions) Facsaria 3, by BD (2 mentions) Facsaria 2, by BD (2 mentions) Anti cd4 alexa fluor 700, by Thermo Fisher Scientific (2 mentions) Lsr 2 device, by BD (2 mentions) Cd25 pc61.5, by Thermo Fisher Scientific (2 mentions)

27 protocols using near ir dead cell stain kit

PBMC Dead Cell Staining and Compensation

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After washing PBMCs with PBS to stop stimulation, they were incubated for 15 min with a Near-IR Dead Cell Stain Kit (Invitrogen). Without washing, cells were stained using an antibody cocktail (Supplementary Table S1) for 20 min at room temperature (RT). After washing with PBS, cells were fixed with 1% PFA (Morphisto GmbH, Offenbach am Main, Germany) for at least 1 h at 4 °C. Washed cells were then stored at 4 °C until flow analysis was performed within 8 h.
Rainbow Calibration Particles (BD Sphero, BD Biosciences, Heidelberg, Germany) were used to ensure the consistency of fluorescence intensities between experiments. Anti-Mouse Ig and k/Negative Control Compensation Particles Set (BD Biosciences) were used for antibody compensation, while ArC Amine Reactive Compensation Beads (Invitrogen) were used to compensate for the Dead Cell Stain Kit as described before.
Flow analysis was performed with a BD FACSymphony A3 flow cytometer in the Cytometry and Cell Sorting Core Unit at the University Medical Center, Eppendorf, Hamburg.
The gating strategy for the AIM+ T cells is depicted in detail in Supplementary Figure S1. The Protocol was previously described in [18 (link)].

Lalia J.K., Schild R., Lütgehetmann M., Dunay G.A., Kallinich T., Kobbe R., Massoud M., Oh J., Pietzsch L., Schulze-Sturm U., Schuetz C., Sibbertsen F., Speth F., Thieme S., Witkowski M., Berner R., Muntau A.C., Gersting S.W., Toepfner N., Pagel J, & Paul K. (2023). Reduced Humoral and Cellular Immune Response to Primary COVID-19 mRNA Vaccination in Kidney Transplanted Children Aged 5–11 Years. Viruses, 15(7), 1553.

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Profiling Human Tumor Immune Cells

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Single cell suspensions from human tumors and peripheral blood were stained with antibodies listed in table 1. Live/dead discrimination was performed using fixable Aqua or Near-IR Dead Cell Stain Kit (Invitrogen). Samples were acquired with a Becton Dickinson LSRII or sorted with a Becton Dickinson FACSAria II and analyzed using FlowJo software. For intracellular staining, cells were fixed and permeabilized using the FOXP3 Transcription Factor Staining Buffer Set (eBioscience). For cell sorting, cryopreserved samples were thawed, stained, and sorted. For RNA and TCR sequencing experiments, total and naïve CD8 T cells were sorted. For TCR sequencing experiments, CD8 T cells were gated as shown, and CD38 +HLA-DR+ were sorted.

Carlisle J.W., Jansen C.S., Cardenas M.A., Sobierajska E., Reyes A.M., Greenwald R., Del Balzo L., Prokhnevska N., Kucuk O., Carthon B.C., Mullane P.C., Osunkoya A., Baumgarten D., Hosseinzadeh F., Wilkinson S., Lake R., Sowalsky A.G., Liu Y., Master V.A., Bilen M.A, & Kissick H. (2022). Clinical outcome following checkpoint therapy in renal cell carcinoma is associated with a burst of activated CD8 T cells in blood. Journal for Immunotherapy of Cancer, 10(7), e004803.

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Comprehensive Immune Cell Phenotyping

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Cells were labelled with combinations of PE-, FITC-, PerCP-, APC-, PE-Cy7-, V450- and Pacific Orange-conjugated mouse anti-human CD14, CD80, CD86, DC-SIGN (CD209) (all from BD Biosciences, Franklin Lakes, NJ, USA), PD-L1 (CD274) (Biolegend) and HLA-DR (Invitrogen) antibodies and appropriate isotype controls, using 0.25 μg of each antibody per staining reaction. Acquisition was performed on a BD FACSCanto II flow cytometer (BD Biosciences) and analysis of flow data was performed using FlowJo 7.6.4 (Tree Star, Inc., Ashland, OR, USA). For viability staining, cells were labelled with a Near-IR dead cell stain kit (Invitrogen) according to the manufacturer’s instructions and analysed by flow cytometry.

Sampangi S., Kassianos A.J., Wang X., Beagley K.W., Klein T., Afrin S., Healy H, & Wilkinson R. (2015). The Mechanisms of Human Renal Epithelial Cell Modulation of Autologous Dendritic Cell Phenotype and Function. PLoS ONE, 10(7), e0134688.

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Phenotyping Human Tumor Cells

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Single cell suspensions from human tumours were stained with antibodies listed in Supplementary Information Table 1. Live/dead discrimination was performed using fixable Near-IR Dead Cell Stain Kit (Invitrogen). Samples were acquired with a Becton Dickinson LSRII and analysed using FlowJo. For intracellular staining, cells were fixed and permeabilized using the FOXP3 Transcription Factor Staining Buffer Set (eBioscience).

Jansen C.S., Prokhnevska N., Master V.A., Sanda M.G., Carlisle J.W., Bilen M.A., Cardenas M., Wilkinson S., Lake R., Sowalsky A.G., Valanparambil R.M., Hudson W.H., McGuire D., Melnick K., Khan A.I., Kim K., Chang Y.M., Kim A., Filson C.P., Alemozaffar M., Osunkoya A.O., Mullane P., Ellis C., Akondy R., Im S.J., Kamphorst A.O., Reyes A., Liu Y, & Kissick H. (2019). An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature, 576(7787), 465-470.

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Clonogenic Survival Assay for LOXPP-Expressing Cells

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Empty vector and rLOX-PP expressing DU145 and PC3 cells were plated and grown in DMEM and 10% serum under standard conditions in 6-well plates. At 80% visual confluence, cells were placed in serum-free medium overnight. Cells expressing rLOX-PP and their respective controls (n=6) were then irradiated and then plated in 6-well plates as follows. Cells were trypsinized and subjected to sorting (Aria FACS, BioRad, Hercules, CA). The cell sorting was performed to exclude dead cells due to IR treatment by utilizing the Near-IR dead cell stain kit (Invitrogen Inc., Carlsbad, CA). The number of cells needed for successful initial plating of 5,000 and 10,000 cells per well in 6-well plates was determined based on the plating efficiency determined at 0 Gy. The clonogenic survival assay, calculation of plating efficiency and the survival fraction determinations were performed as described (62 (link), 63 (link)). Colonies were allowed to grow for 14 days, and cultures were then fixed, and stained with crystal violet and colony numbers were determined using a Versadoc Photodocumentation System and Quantity One colony counting software (BioRad, Hercules, CA). The survival fraction was calculated and plotted as described (62 (link)).

Bais M.V., Ozdener G.B., Sonenshein G.E, & Trackman P.C. (2014). Effects of Tumor Suppressor Lysyl Oxidase Propeptide on Prostate Cancer Xenograft Growth and Its Direct Interactions with DNA Repair Pathways. Oncogene, 34(15), 1928-1937.

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Isolation of Naïve Tconv and tTreg Cells

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Human materials were obtained in compliance with the Declaration of Helsinki and the Dutch rules regarding the use of human materials from voluntary donors. Buffy coats from healthy anonymized male donors were obtained after their written informed consent, as approved by the Ethical Advisory Council of Sanquin Blood Supply Foundation, Plesmanlaan 125, Amsterdam, the Netherlands. Total CD4⁺ T cells were collected from buffy coats by first performing a Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation to isolate PBMCs and subsequently using CD4 magnetic MicroBeads (Miltenyi Biotec) according to the manufacturers’ protocols. Alternatively, total CD4⁺ T cells were directly isolated from buffy coats using the StraightFrom Buffy Coat CD4 MicroBead kit (Miltenyi Biotec) according to the manufacturer’s instructions. To sort naïve Tconv and tTreg cells, CD4⁺ T cells were stained with CD4-PE-Cy7, CD127-BV421 (BioLegend), CD25-PE (BD Biosciences), CD45RA-FITC (Immunotools) and GPA33-AF647^{68 (link)} mAbs as indicated (Supplementary Fig. 1). Cells were sorted on a MoFlo Astrios using Summit software version 6.2 (Beckman Coulter) or BD FACS Aria II using FACSDiva software version 8 (BD Biosciences). Either propidium iodide (Sigma) or near-IR dead cell stain kit (Invitrogen) was used as a live/dead marker.

Mensink M., Schrama E., Cuadrado E., Amsen D., de Kivit S, & Borst J. (2022). Proteomics reveals unique identities of human TGF-β-induced and thymus-derived CD4+ regulatory T cells. Scientific Reports, 12, 20268.

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Isolation and Characterization of moDCs

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For in vitro DC-T cell co-culture, bulk mRNA-Seq and CITE-Seq experiments, PBMCs were directly used for FACS. For ex vivo DC-CD4⁺ T-cell co-culture experiments, CD19⁺ cells were depleted before sorting using CD19 magnetic MicroBeads (MACS), according to the manufacturer’s protocol. Staining was performed at 4 °C for 45 min in flow cytometry staining buffer (BD Biosciences). The following antibodies were used: from BioLegend: CD1c (clone L161), CD3 (clone OKT3), CD4 (clone OKT4), CD8 (clone SK1), CD11c (clone Bu15/3.9), CD14 (clone M5E2), CD19 (clone HIB19), CD25 (BC96), CD45RA (clone HI100), CD141 (clone M80), CD206 (clone 15-2), CD303 (clone 201 A), HLA-DR (clone L243); from BD Biosciences: HLA-DR (clone G46-6); from Miltenyi Biotec: CD141 (clone REA674). Near-IR Dead Cell Stain Kit (Invitrogen), Zombie Red Fixable Viability Kit (BioLegend) or 7-amino-actinomycin D (7-AAD, eBioscience) were used to exclude dead cells. In each sorting, cDC2 (within the same sample) was used as a negative control for gating ex vivo moDC. Detailed information regarding these antibodies can be found in the Reporting Summary. In order to prevent clump formation from dead cells, 0.01% DNase (Invitrogen) was added before sorting. Cell sorting was performed on BD FACSAria^TM Fusion or BD FACSAria III (BD Biosciences).

Lei X., Khatri I., de Wit T., de Rink I., Nieuwland M., Kerkhoven R., van Eenennaam H., Sun C., Garg A.D., Borst J, & Xiao Y. (2023). CD4+ helper T cells endow cDC1 with cancer-impeding functions in the human tumor micro-environment. Nature Communications, 14, 217.

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Multiparameter PBMC Profiling by FACS

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For in vitro DC-T-cell coculture and NanoString nCounter gene expression analysis, PBMCs were directly used for FACS. CD19⁺ cells were depleted before sorting using CD19 magnetic MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol. Staining was performed at 4 °C for 45 min in flow cytometry staining buffer (BD Biosciences). Antibodies specific for the following proteins were used: from BioLegend, CD1c (PE-Cy7, clone L161), CD3 (BV510, clone OKT3), CD4 (FITC, clone OKT4), CD8 (Percp-cy5.5, clone SK1), CD11c (PE/AF700, clone Bu15/3.9), CD14 (ef450, clone M5E2), CD19 (BV510, clone HIB19), CD25 (PE, clone BC96), CD45RA (AF700, clone HI100), CD141 (BV421, clone M80), and HLA-DR (BV605, clone L243); from BD Biosciences, HLA-DR (APC-Cy7, clone G46-6); and from Miltenyi Biotec, CD141 (APC, clone REA674). A Near-IR Dead Cell Stain Kit (Invitrogen), a Zombie Red Fixable Viability Kit (BioLegend) or 7-aminoactinomycin D (7-AAD, eBioscience) was used to exclude dead cells. To prevent clumping of dead cells, DNase I (0.1 mg/ml) was added before sorting. Cell sorting was performed on a BD FACSAria III (BD Biosciences).

Lei X., de Groot D.C., Welters M.J., de Wit T., Schrama E., van Eenennaam H., Santegoets S.J., Oosenbrug T., van der Veen A., Vos J.L., Zuur C.L., de Miranda N.F., Jacobs H., van der Burg S.H., Borst J, & Xiao Y. (2024). CD4+ T cells produce IFN-I to license cDC1s for induction of cytotoxic T-cell activity in human tumors. Cellular and Molecular Immunology, 21(4), 374-392.

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Clonogenic Survival Assay for LOXPP-Expressing Cells

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Flow Cytometric Analysis of Murine Leukocytes

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Flow cytometric analyses were conducted on a FACSCanto II or LSR II (BD Biosciences, Franklin Lakes, NJ, USA). All antibodies were from BioLegend or eBioscience (Thermo Fisher Scientific, Bremen, Germany), unless otherwise stated. LIVE/DEAD Fixable Violet or Near-IR Dead Cell Stain Kit (Invitrogen, Thermo Fisher Scientific) was used to exclude dead cells in all samples analysed. Anti-CD16/32 antibody (clone 2.4G2) was included in each staining at 10 μg/mL to block unspecific antibody binding via Fc receptors. Leukocytes were stained with antibodies against murine CD11b (clone M1/70), CD11c (clone N418), SiglecF (clone E50-2440), Ly6G (clone 1A8), Ly6C (clone HK1.4), CD64 (clone X54-5/7.1) and Clec2a (clone 17D9). Data were analysed using FlowJo software version 10 (Becton, Dickinson & Company, Ashland, OR, USA).

Unrau L., Endig J., Goltz D., Sprezyna P., Ulrich H., Hagenstein J., Geers B., Kaftan K., Heukamp L.C., Tiegs G, & Diehl L. (2021). Smad7 Deficiency in Myeloid Cells Does Not Affect Liver Injury, Inflammation or Fibrosis after Chronic CCl4 Exposure in Mice. International Journal of Molecular Sciences, 22(21), 11575.

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