Live dead fixable blue dead cell stain kit for uv excitation

Manufactured by Thermo Fisher Scientific

The LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation is a fluorescent dye-based reagent used to distinguish between live and dead cells. The dye binds to proteins in dead cells, emitting a fluorescent signal that can be detected using flow cytometry or fluorescence microscopy. The kit is designed for use with UV excitation.

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10 protocols using live dead fixable blue dead cell stain kit for uv excitation

Murine Immune Cell Phenotyping

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Antibodies to mouse CD45 (clone 30‐F11), CD19 (clone 6D5), NK‐1.1 (clone PK136), CD4 (clone RM4‐5), CD90.2 (clone 30‐H12), CD3e (clone 145‐2C11), CD11b (clone M1/70), CD11c (clone N418), ICOS (clone7E.17G9), IL‐5 (clone TRFK5), IL‐13 (clone eBio13A), Ki‐67 (clone 16A8) and Ly6G (clone 1A8) were purchased from eBioscience or Biolegend. Antibodies to mouse ST2(IL33‐R) (clone DJ8) and SiglecF (clone E50‐2440) were purchased from mdbioproducts and BD respectively. LIVE/DEAD™️ Fixable Blue Dead Cell Stain Kit for UV excitation, UltraComp eBeads™️ and mouse IL‐5/IL‐13 ELISA kits were purchased from Thermo Fisher Scientific. Recombinant mouse IL‐33 were purchased from Biolegend. PGE₂, Butaprost (free acid) and L‐902688 (EP4 agonist) were obtained from Cayman, while PF‐04418948 was purchased from Abcam. Indomethacin, phorbol myristate acetate (PMA), ionomycin, brefeldin A, dibutyryl cyclic adenosine monophosphate (db‐cAMP) and 3‐isobutyl‐1‐methyxanthine (IBMX) were purchased from Sigma. Complete RMPI consisted of RPMI 1640 (Gibco) supplemented with 10% FBS, 1% Penicillin/Streptomycin, 1% L. glutamine and 50 μM β‐mercaptoethanol.

Robb C.T., Zhou Y., Felton J.M., Zhang B., Goepp M., Jheeta P., Smyth D.J., Duffin R., Vermeren S., Breyer R.M., Narumiya S., McSorley H.J., Maizels R.M., Schwarze J.K., Rossi A.G, & Yao C. (2022). Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses. Allergy, 78(3), 714-730.

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Multiparametric Flow Cytometric Profiling of Liver, Spleen, and PB NK Cells

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In order to compare the phenotype of liver, spleen, and PB NK cells, we performed multiparametric flow cytometry. Lymphocytes harvested from fetal liver, fetal spleen, adult liver, adult spleen, and PB were freshly stained ex vivo. Cells were counted, and viability was assessed by trypan blue before staining. 10⁶ cells in 100 μL PBS were aliquoted into 5 mL FACS tubes. LIVE/DEAD fixable blue dead cell stain kit for UV excitation (ThermoFisher) was added for 7–10 min in the dark at RT. Extracellular antibodies were added for 20 min at RT (see Table S1 for all antibodies used in this study). For intranuclear staining of Tbet and EOMES, cells were washed with 2 mL of FACs buffer after extracellular antibodies, then resuspended in 1 mL of TONBO FOXp3/Transcription Factor Fix/Perm buffer (TONBO Biosciences) for 30 min at RT. After permeabilization, cells were washed with 2 mL of TONBO wash buffer. Intra-nuclear antibodies (anti-Tbet-BV711 and anti-EOMES-PerCP/Cy5.5) were added and incubated for 30 min at RT. Cells were washed with TONBO wash buffer, then FACS buffer and resuspended in a final concentration of 1% paraformaldehyde (PFA) and kept at 4°C in the dark until acquisition on a BD LSR Fortessa with 18 color configuration.

Angelo L.S., Bimler L.H., Nikzad R., Aviles-Padilla K, & Paust S. (2019). CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities. Frontiers in Immunology, 10, 469.

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Comprehensive Immune Profiling of PBMCs

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Peripheral blood samples were first stained for surface epitopes [αCD8 AF700 (53-6.7), αCD28 BV510 (CD28.2), αCD69 FITC (FN50), αCD160 PE-Cy7 (By55), αPD-1 PerCP-Cy5.5 (EH12.2H7) from BioLegend] including a live/dead staining reagent (LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation from ThermoFisherScientific) for 30 min at 4°C. Afterwards, lymphocytes were fixed and red blood cells (RBCs) were lysed (1-step Fix/Lyse solution from eBioscience). Fixation and permeabilisation of cells was performed using the Foxp3/Transcription Factor Buffer Set (ThermoFisherScientific) according to the manual. To block unspecific binding of antibodies, cells were incubated for 10 min at 4°C with CohnII and subsequently, antibodies directed against intracellular epitopes (GzmB AF647 (GB11), CTLA-4 PE (L3D10), Ki67 AF488 (Ki-67), CD3 APC-Cy7 (HIT3a) from BioLegend, Perforin BV421 (δG9) from BD) were added and further incubated for 20 min at 4°C. Samples were recorded using the LSRII (BD) and analyzed using the FlowJo X 10.0.7r2 Treestar software. Gates were set according to fluorescence minus one (FMO) controls. Gating strategy is depicted in Supplementary Figure 5.

Muscate F., Stetter N., Schramm C., Schulze zur Wiesch J., Bosurgi L, & Jacobs T. (2018). HVEM and CD160: Regulators of Immunopathology During Malaria Blood-Stage. Frontiers in Immunology, 9, 2611.

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Intracellular Cytokine Staining Protocol

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For intracellular cytokine staining, cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 hours. Brefeldin A 1000X (eBioscience) was added for the final 4.5 hours. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4°C. Cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRp (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), IFNγ (eFluor450; eBioscience), and/or FOXP3 (eFluor450, eFluor660; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells.

O’Driscoll C.A., Owens L.A., Hoffmann E.J., Gallo M.E., Afrazi A., Han M., Fechner J.H., Schauer J.J., Bradfield C.A, & Mezrich J.D. (2018). Ambient urban dust particulate matter reduces pathologic T cells in the CNS and severity of EAE. Environmental research, 168, 178-192.

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Characterizing Recombinant CSP Antibodies

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CSP specific monoclonal antibodies used to characterize recombinant CSP were generated in mice against a full-length CSP recombinant protein [36] (link) and showed positive reactivity to Pf sporozoites by IFA and western blot (Dr. Ted B. Hall, personal communication). PE-anti-mouse IgG, PE-anti-mouse IgG1, PE-anti-mouse IgG2c and PE-anti-mouse IgG2a were obtained from Jackson Immunoresearch (West Grove, PA). Luminex beads and Luminex Streptavidin beads were purchased from Luminex Corporation (Austin, TX) and 96-well Luminex Multi-screen BV 1.2 micron assay plates were acquired from Millipore Corporation (Billerica, MA). Antigens were coupled to the Luminex beads according to the manufacturer’s instructions. PerCP anti-CD3 (clone 145-2C11), Pacific Blue anti-CD4 (clone RM4-5), Horizon V500 anti-CD8 (clone 53-6.7), Alexa 700 anti-CD44 (clone IM7), FITC anti-IL-2 (clone JES6-5H4), APC anti-IFN-γ (clone XMG1.2), anti-CD16/CD32 FcR Block (clone 2.4G2), anti-CD28 (clone 37.51), anti-CD49d [clone 9C10(MFR4.B)], Cytofix/Cytoperm and Golgi Plug were all obtained from BD Biosciences (San Jose, CA). LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation was purchased from Invitrogen (Camarillo, CA). Phorbol 12-myristate 13 acetate (PMA) and Ionomycin were obtained from Sigma Chemicals (St. Louis, MO).

Schwenk R., DeBot M., Porter M., Nikki J., Rein L., Spaccapelo R., Crisanti A., Wightman P.D., Ockenhouse C.F, & Dutta S. (2014). IgG2 Antibodies against a Clinical Grade Plasmodium falciparum CSP Vaccine Antigen Associate with Protection against Transgenic Sporozoite Challenge in Mice. PLoS ONE, 9(10), e111020.

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Intracellular Cytokine Staining of T Cells

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Intracellular cytokine staining was conducted on day 3, after the T cells were cultured for 72 h. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 h. Brefeldin A 1000X (eBioscience) was added for the final 4.5 h. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4 °C. Cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), IFNγ (eFluor450; eBioscience), and/or FOXP3 (eFluor450, eFluor660; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells.

O’Driscoll C.A., Owens L.A., Gallo M.E., Hoffmann E.J., Afrazi A., Han M., Fechner J.H., Schauer J.J., Bradfield C.A, & Mezrich J.D. (2018). Differential effects of diesel exhaust particles on T cell differentiation and autoimmune disease. Particle and Fibre Toxicology, 15, 35.

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Intracellular Cytokine Staining for T Cell Activation

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Intracellular cytokine staining for T cells was conducted on day 3, after the T cells were cultured for 72 h. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 h. Brefeldin A 1000X (eBioscience) was added for the final 4.5 h. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4 °C T cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5, FITC; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17 A (FITC or PE; eBioscience), and/or KI67 (PE; eBioscience). Stained cells were analyzed on Fortessa (BD), Attune NxT (Invitrogen), or Accuri C6 (BD). Data was analyzed using FlowJo software (TreeStar). Flow plots showing percent live cells are showing the percent live cells of total lymphocytes. Flow plots show IL-17 producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells or TCRβ⁺ Live cells.

O’Driscoll C.A., Gallo M.E., Fechner J.H., Schauer J.J, & Mezrich J.D. (2019). Real-world PM extracts differentially enhance Th17 differentiation and activate the aryl hydrocarbon receptor (AHR). Toxicology, 414, 14-26.

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Intracellular Cytokine Staining for T Cells

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Intracellular cytokine staining for T cells was conducted on day 3, after the T cells were cultured for 72 hours. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 hours. Brefeldin A 1000X (eBioscience) was added for the final 4.5 hours. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4°C. T cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), KI67 (PE; eBioscience), or FOXP3 (eFluor450; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells or TCRβ⁺ Live cells.

O’Driscoll C.A., Gallo M.E., Hoffmann E.J., Fechner J.H., Schauer J.J., Bradfield C.A, & Mezrich J.D. (2018). Polycyclic aromatic hydrocarbons (PAHs) present in ambient urban dust drive proinflammatory T cell and dendritic cell responses via the aryl hydrocarbon receptor (AHR) in vitro. PLoS ONE, 13(12), e0209690.

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Analyzing Viral Infection by Flow Cytometry

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RVFV MP12^GFP and RSV^GFP infected cells were analyzed by flow cytometry (BD LSR Fortessa™ X-20 and BD LSR Fortessa™) and the data were analyzed using BD FACS Diva software, as described previously (Ganaie et al., 2017 (link)). All flow experiments were done at the Flow Cytometry Facility, Department of Pathology and Immunology, WUSM. For flow experiments with RVFV ZH501, infected cells were harvested at the indicated time points, stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit for UV excitation (Invitrogen L34961), permeabilized with BD fix/perm, then stained with RVFV anti-Gn monoclonal antibody (BEI NR-43195) followed by a FITC-conjugated anti-mouse secondary antibody. Samples were acquired using BD LSRII flow cytometer and analyzed with FlowJo at the University of Pittsburgh Flow core facility. Uninfected cells were run in parallel for subtraction of background.

Ganaie S.S., Schwarz M.M., McMillen C.M., Price D., Feng A., Albe J.R., Wang W., Miersch S., Orvedahl A., Cole A.R., Sentmanat M.F., Mishra N., Boyles D.A., Koenig Z.T., Kujawa M.R., Demers M.A., Hoehl R.M., Moyle A., Wagner N., Stubbs S.H., Cardarelli L., Teyra J., McElroy A., Gross M.L., Whelan S.P., Doench J., Cui X., Brett T.J., Sidhu S.S., Virgin H.W., Egawa T., Leung D.W., Amarasinghe G.K, & Hartman A.L. (2021). Lrp1 is a host entry factor for Rift Valley Fever Virus. Cell, 184(20), 5163-5178.e24.

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Intracellular Cytokine Profiling of BMDCs

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Intracellular cytokine staining for BMDCs was conducted on day 6, 24 hours after treatment. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 hours. Brefeldin A 1000X (eBioscience) was added for the final 4.5 hours. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) and intracellular cytokines overnight at 4°C. BMDCs were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD11c (PeCy7; eBioscience), F4/80 (PE; eBioscience), CD86 (APC; eBioscience), and MHC II (BV605; eBioscience) for extracellular markers. Cells were stained with IFNγ (eFluor488; eBioscience) and IL-10 (AlexaFluor700). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD11c⁺F4/80^- Live cells.

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