Cells were washed and stained with the following fluorescent antibodies: PE anti-human PD-L1 (#12-5983-42, eBioscience), PE anti-mouse PD-L1 (#124308, BioLegend) and APC anti-human MHC class I (#311409, BioLegend). After incubation, the cells were washed with PBS containing 0.5% BSA and run on a BD FACSVerse flow cytometer (BD Biosciences). A LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit (#L34974, Invitrogen) was used to gate live cells. The results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). For flow cytometric analysis of PD-L1 induced by IFN-γ, cells were treated with 10 ng/ml human IFN-γ (#PHC4031, Gibco) for 24 h.
Live dead fixable far red dead cell stain kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Canada
The LIVE/DEAD Fixable Far Red Dead Cell Stain Kit is a laboratory product designed to detect and distinguish between live and dead cells. It utilizes a fluorescent dye that binds to dead cell proteins, allowing for their identification through flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Facsdiva software, by BD (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Ldh glo cytotoxicity assay, by Promega (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Pe anti mouse pd l1, by BioLegend (1 mentions)
Pe cy5 anti mouse cd8a, by BD (1 mentions)
Facsverse cytometer, by BD (1 mentions)
Accuri flow cytometer, by BD (1 mentions)
35 μm preseparation filter, by Corning (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Nacalai Tesque (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
50 protocols using live dead fixable far red dead cell stain kit
1
Flow Cytometric Analysis of PD-L1 Expression
2
Quantifying Virus-specific T-cells in Murine Splenocytes
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Spleens from mice immunized with either LAIV H1N1 6 : 2 or LAIV H1N1 5 : 3, and from mock-immunized mice, were collected on days 21 and 42 after inoculation. Single spleen cell suspensions were prepared and frozen in liquid nitrogen until the assay. Virus-specific CD4+ and CD8+ Т-lymphocytes were determined by flow cytometry. Virus-specific T-cells were identified by conventional intracellular cytokine (gamma interferon) staining. Briefly, the 1 × 106 of splenocytes were stimulated in vitro with SA/wt virus at 1.5 MOI in RPMI-1640 medium not containing fetal bovine serum. After 1-hour stimulation, fetal bovine serum was added to give 10% final concentration and cells were further incubated overnight. As a negative control, spontaneous gamma interferon production was determined by adding the appropriate volume of RPMI-1640 nutrient medium to cells; the resulting measurement was subtracted from the data obtained for virus-stimulated cells. The following monoclonal antibodies were used for staining: (1) Live/Dead Fixable Far Red Dead Cell Stain Kit (Invitrogen); (2) PE-Cy5 Anti-Mouse CD8a (BD Pharmingen); (3) Rat Anti-Mouse CD4/L3T4a-FITC (Beckman Coulter); and (4) PE Rat Anti-Mouse Gamma Interferon (BD Pharmingen). CD4+IFNγ+ and CD8+IFNγ+ T-cell populations were analyzed within live cells gate.
3
Tumor Immune Profiling by Flow Cytometry
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After euthanasia, tumors were surgically harvested and disassociated into single cells over a 40-μM mesh filter. Single cell suspensions were then incubated with the indicated antibodies, washed with PBS, and stained with the LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) using standard flow cytometry methodologies. Samples were then analyzed on a BD FACSVerse cytometer (BD Biosciences, San Jose, CA, USA). All analyses shown are pregated on single, viable events. Immune populations were defined as follows: CD8+ T cells (CD45+/CD3+/CD8+/CD4−), conventional CD4+ T cells (Tcon; CD45+/CD3+/CD8−/CD4+), MACS (CD45+/F4/80+),32 (link) plasmacytoid myeloid-derived suppressor cells (pMDSCs; CD45+/F4/80−/Ly6Chi/Ly6G−), and monocytic MDSCs (mMDSCs; CD45+/F4/80−/Ly6Clo/Ly6G+).33 (link)
4
Cytotoxicity Assay for Compounds
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The cytotoxicity of compounds was determined by using the ATP-based CellTiter-Glo Luminescent Cell Viability Assay (Promega) and analyzed by the Cytation 5 multimode reader (luminescent mode). CC50 was plotted and calculated by using the GraphPad Prism 6.0 software. Death of virus-infected or compound-treated cells was determined by using the LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen) and analyzed by the BD Accuri C6 Plus (flow cytometry).
5
Measuring Retinal ROS Production in Lpcat1 Mice
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ROS production was measured by flow cytometry using MitoSOX Red (Invitrogen). Retina was isolated from 3-week-old Lpcat1 WT and KO mice and dissociated in 500 μl of 0.25% trypsin (Nacalai Tesque) in PBS for 15 min at 37 °C. Then, 500 μl of 20% fetal bovine serum (Thermo Fisher Scientific) and 1 μl DNase I (Invitrogen) were added on ice. Mechanical dissociation was performed by pipetting 20 times using a 1-ml tip. The cells were collected by centrifugation at 300g for 5 min. The retinal cell pellet was washed in 700 μl of 2% BSA/PBS and centrifuged at 300g for 5 min. The cell pellets were resuspended in 2% BSA/PBS. Photoreceptor cells were labeled with FITC anti-mouse CD73 antibody (TY/11.8, BioLegend). Dead cells were stained with LIVE/DEAD Fixable Far Red Dead Cell Stain kit (Invitrogen), and retinal cells were stained with 5 μM MitoSOX Red for 15 min at 37 °C. The cells were centrifuged at 300g for 5 min. The cell pellet was resuspended in 500 μl of 2% BSA/PBS and filtered through a 35-μm preseparation filter (Corning). The fluorescence intensity was measured using a BD Accuri flow cytometer (BD Biosciences).
6
Cellular Viability Assessed by Flow Cytometry
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Cellular viability was determined by flow cytometry using LIVE/DEAD Fixable Far Red Dead Cell Stain kit (Invitrogen™, Thermo Fisher Scientific) according to manufacturer’s instructions by samples acquisition on a CYTOMICS FC500 flow-cytometer. Data were analysed as % of dim or bright positive cells by Kaluza software (Beckman Coulter).
7
Evaluating 25-HC@DDAB Cytotoxicity
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Mouse lung endothelial cells were treated with various concentrations of 25-HC@DDAB for 24 h and stained using LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit (L34973, Invitrogen™). Cells were then washed twice with PBS. Stained cells were resuspended in 1 mL of PBS, and the fluorescence was quantified using a FACScan flow cytometer (BD).
8
Cytotoxicity Assay for Compound Screening
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The cytotoxicity of compounds was determined by using the ATP- based CellTiter-Glo Luminescent Cell Viability Assay (Promega) and analyzed by the Cytation 5 multimode reader (luminescent mode). Death of virus-infected or compound-treated cells was determined by using the LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen) and analyzed by the BD Accuri C6 Plus (flow cytometry) [15 (link)].
9
Melanoma Cell Viability Assessment
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In order to assess the viability of the cells grown in the three described conditions, cells from three melanoma cell lines (MML‐1, UM22Bap1+/+ and UM22Bap1–/–) were seeded in flat‐bottomed 6‐well plates and incubated at 37°C with 5% CO2 for 72 h. The cell number was determined manually using a trypan blue exclusion assay and an optical microscope, and 5 × 105 cells/well were seeded in complete medium containing FBS processed by one of the three protocols. After 72 h, the cells were photographed using the EVOS XL Core Imaging System (Life Technologies, Bothell, WA, USA). The cells were gently detached from the cell culture plate with 0.25% trypsin at 37°C for approximately 3 min. After washing, one aliquot of each cell line was used for manual counting of the cell number, while the rest of the cells were stained with a Live/Dead Fixable Far Red Dead Cell Stain Kit (Invitrogen, Oregon, US) in phosphate‐buffered saline (PBS) and analysed on a BD LSRFortessa instrument (BD Biosciences, San Jose, CA). Subsequent data analysis was performed in BD FACSDiva Software version 9.0.1 (BD Biosciences).
For doubling time calculations, we used the following formulation:
where the duration was 72 h.
For doubling time calculations, we used the following formulation:
where the duration was 72 h.
10
Quantification of Lung Apoptosis
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Quantification of the overall lung caspase-3 activity in lung homogenates and caspase-3 immunohistochemistry on lung sections was performed as described previously (Bem et al. 2010c (link)). To quantify lung epithelial cell apoptosis by immunohistochemistry, the number of cleaved caspase-3-positive epithelial cells (airway and alveolar) was counted in four random high-power fields per lung tissue section.
To obtain single lung cell suspensions for FACS analysis, lungs were perfused with 20 mL PBS through the right ventricle, minced using iridectomy scissors, and digested with collagenase III. Finally, the cells were passed through a 70-μm cell strainer and subjected to RBC lysis, and kept on ice until labeling. The isolated cells from the lung and BALF were Fc-blocked with antimurine CD16/CD32 and labeled with: Annexin V-APC (A35110; Life-Technologies-Invitrogen, Bleiswijk, the Netherlands), LIVE/DEAD fixable far red dead cell stain kit (Life-Technologies-Invitrogen, Bleiswijk and the Netherlands) and the neutrophil marker Ly-G6 (GR1-PE; eBioscience, Hatfield, UK). Annexin V-positive and LIVE/DEAD far red stain-negative cells were considered apoptotic.
To obtain single lung cell suspensions for FACS analysis, lungs were perfused with 20 mL PBS through the right ventricle, minced using iridectomy scissors, and digested with collagenase III. Finally, the cells were passed through a 70-μm cell strainer and subjected to RBC lysis, and kept on ice until labeling. The isolated cells from the lung and BALF were Fc-blocked with antimurine CD16/CD32 and labeled with: Annexin V-APC (A35110; Life-Technologies-Invitrogen, Bleiswijk, the Netherlands), LIVE/DEAD fixable far red dead cell stain kit (Life-Technologies-Invitrogen, Bleiswijk and the Netherlands) and the neutrophil marker Ly-G6 (GR1-PE; eBioscience, Hatfield, UK). Annexin V-positive and LIVE/DEAD far red stain-negative cells were considered apoptotic.
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