Fmoc-protected amino acids, resins, and coupling reagents for solid-phase synthesis were purchased from EMD Millipore. Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) was purchased from Sigma-Aldrich. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-mPEG1000 (mPEG1000-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000-azide (N3-PEG2000-DSPE), and cholesterol (Chol) were purchased from Avanti Polar Lipids. Cyanine 5.5-NHS ester was purchased from Lumiprobe. Cellulose acetate (CA) syringe filters were purchased from Macherey-Nagel Inc. Sephadex® G-50 and LH-20 were purchased from GE Healthcare. LIVE/DEAD® fixable green dead cell stain was purchased from ThermoFisher. AlexaFluor® 680-Wheat Germ Agglutinin conjugate (WGA) and LysoTracker® Blue DND-22 were purchased from Life Technologies. Cell culture reagents such as DMEM, FBS, trypsin, and PBS were purchased from Atlanta Biologicals. Ultrapure water (18 MΩ) was used for preparation of all buffers and in all experiments. All solvents were of analytical grade, purchased from commercial sources and used without further purification, except DMF which was dried over CaH2 under N2, filtered, and distilled under reduced pressure.
Live dead fixable green dead cell stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD™ Fixable Green Dead Cell Stain is a fluorescent dye used to detect dead cells in a sample. It binds to proteins in dead cells, allowing them to be identified and quantified.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (3 mentions)
Red blood cell lysis buffer, by Merck Group (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Golgiplug, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Trypsin, by Atlanta Biologicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Atlanta Biologicals (1 mentions)
Lh 20, by GE Healthcare (1 mentions)
Lysotracker blue dnd 22, by Thermo Fisher Scientific (1 mentions)
Tris 3 hydroxypropyltriazolylmethyl amine thpta, by Merck Group (1 mentions)
Sephadex g 50, by GE Healthcare (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Cholesterol chol, by Avanti Polar Lipids (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Fmoc protected amino acids, by Merck Group (1 mentions)
Cyanine5.5 nhs ester, by Lumiprobe (1 mentions)
Il 2 pe, by BD (1 mentions)
Ifn γ alexa647, by BD (1 mentions)
Cd8 percp, by BD (1 mentions)
17 protocols using live dead fixable green dead cell stain
1
Solid-Phase Synthesis of Lipid Nanoparticles
2
Single-Cell RNA-Seq of Tumor Subpopulations
3
Evaluation of ZIKV NS1 Cellular Immune Response
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CD-1/ICR mice immunized once with 1 × 107 TCID50 MVA-ZIKV-NS1 vaccine were sacrificed 10 days after immunization, and spleens were harvested. Naïve mouse spleens were harvested in parallel. Isolated splenocytes were analyzed by intracellular cytokine staining following standard protocols. In short, 106 splenocytes were stimulated with 0.1 µg ZIKV NS1 peptide library (JPT Peptide Technology), 0.1 µg HIV Env peptide library (21st Century Biochemicals) or mock stimulated. Incubation proceeded for 6 hours at 37° C +5% CO2 and GolgiPlug (BD Biosciences) was added at a concentration of 1.0 µL/mL for the last 4 hours of the incubation. The splenocytes were stained with Live/Dead Fixable Green Dead Cell Stain (ThermoFisher) at room temperature for 20 minutes. The samples were treated with Cytofix/Cytoperm (BD Biosciences) at 4° C for 20 minutes and were stained with CD3 APC-CY7, CD4 PE-Cy7, CD8 PerCP, IL2 PE, and IFN-γ Alexa647 (all flow cytometry antibodies from BD Biosciences). Samples were analyzed with a FACSCanto flow cytometer (BD Biosciences) utilizing FACSDiva software. Analysis was performed with FlowJo software. Cytokine responses were scored as a percent of total CD4+ (Fig. 4e ) or CD8+ (Fig. 4f ) T cells.
4
ATRA and TCP Induce Cell Differentiation
5
Tumor Cell Isolation and Single-Cell RNA-Seq Analysis
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Following visualization with NIR microscopy, sections of tumor that were positive for HS196 signal were dissected and enzymatically digested. Red blood cells were lysed with Red Blood Cell Lysis buffer (Sigma R7757) for 5 minutes, and stained with LIVE/DEAD Fixable Green Dead Cell Stain (Thermo Fisher). Live, single cells from the tumor suspension were sorted by flow cytometry into HS196+ (top 2% of cells) and HS196− (bottom 25% of cells) portions. 10× libraries were created using Chromium Single-Cell 5′ Library Construction Kits (v1.1; 10X Genomics) following the manufacturer's protocol. A targeted cell recovery of 7,000 cells was used for each tumor sample. Gene-expression libraries were created for each sample. Generated cDNA and final GEX libraries were quality checked using an Agilent Bioanalyzer 2100 at Duke Microbiome Core Facility and submitted to MedGenome Inc. for sequencing on a NovaSeq S4 instrument. All RNA-seq data have been deposited in the NCBI GEO and are available under the accession number GSE164865.
6
Lysosomal Integrity Assay Using LysoTracker
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1E06 cells were stained with 50 nm of lysosomotropic dye (LysoTracker Red DND-99, L7528, Thermo Fisher Scientific) and LIVE/DEAD™ Fixable Green Dead Cell Stain (L34969, Thermo Fischer Scientific) for 15 min. Positive control conditions were pre-incubated with 1 mM LLOMe (L7393, Sigma) for 10 min at 37 °C and STING inhibition conditions with 1 μM H151 (941987-60-6, Sanbio) for 5 h. The MFI was calculated and the leakiness propensity was calculated as the +LLOMe/untreated ratio. For Gal3 staining, see the confocal microscopy section.
7
Bacterial Strain Preparation and Characterization
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Human tubal fluid (HTF) medium was purchased from Biocare Europe (Rome, Italy). MitoSOX™ Red, LIVE/DEAD™ Fixable Green Dead Cell Stain and Vybrant FAM Caspase 3 and 7 Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). In Situ Cell Death Detection Kit, fluorescein was obtained from Sigma Aldrich (St. Louis, MO, USA).
Seven bacterial reference strains were included in the study as follows: two isolates of E. coli (ATCC 29522 and ATCC 35218), one isolate of K. pneumoniae (ATCC 13883), one isolate of K. quasipneumoniae (ATCC 700603), one isolate of P. aeruginosa (ATCC 27853), one E. cloacae (ATCC 13047) and one K. aerogenes (ATCC 13048). An E. coli K12 strain MG1655 and its derivative (with the knockout recX gene), from the Keio collection, were also added to the study collection52 (link). Most of the selected reference strains were isolated from clinical human samples, except for K. pneumoniae ATCC 13883 and E. coli ATCC 25922 whose source is unknown (Supplemental TableS1 ). All bacterial strains were seeded on CHROMID® CPS® Elite agar (bioMérieux, Marcy l’Etoile, France) and incubated for 18 h at 35 ± 1 °C. Bacterial suspensions were prepared in 2 ml of sterile water and optical density was measured by DensiCHEK™ spectrophotometer (bioMérieux).
Seven bacterial reference strains were included in the study as follows: two isolates of E. coli (ATCC 29522 and ATCC 35218), one isolate of K. pneumoniae (ATCC 13883), one isolate of K. quasipneumoniae (ATCC 700603), one isolate of P. aeruginosa (ATCC 27853), one E. cloacae (ATCC 13047) and one K. aerogenes (ATCC 13048). An E. coli K12 strain MG1655 and its derivative (with the knockout recX gene), from the Keio collection, were also added to the study collection52 (link). Most of the selected reference strains were isolated from clinical human samples, except for K. pneumoniae ATCC 13883 and E. coli ATCC 25922 whose source is unknown (Supplemental Table
8
Isolation and Analysis of PBMCs
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Peripheral blood mononuclear cells were isolated by Ficoll-Paque (GE Lifesciences) gradient centrifugation. The cells were frozen at 21408C after isolation with CTL-Cryo ABC (Cellular Technologies Limited [CTL]) freezing kit and thawed with RPMI 1640 with CTL Wash (CTL). Staining was performed at 148C for 30 minutes with antibodies and with Live/Dead Fixable Green Dead Cell Stain (at a dilution of 1:500; Thermo Fisher Scientific) diluted in Brilliant Stain Buffer (BD Biosciences). For detection of FOXP3, the cells were permeabilized after surface staining with Foxp3 transcription factor staining kit (eBioscience). Flow cytometry data were acquired with a LSR Fortessa device (BD Bioscience) and analyzed by FlowJo software (BD Bioscience). The gating was determined using biological negative populations. The antibodies used in the study are listed in Table E2 in the Online Repository available at www. jacionline.org.
9
Multiparametric Flow Cytometry Staining
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For staining of surface antigens fresh or thawed cells were incubated 30 minutes at +4c with antibodies and with Live/dead Fixable Green Dead Cell Stain (at dilution of 1:500; ThermoFisher) that were diluted in in Brilliant Stain Buffer (BD Bicoscience). When applicable the cells were incubated with MR-1 tetramer (1:2500) in +37c for 45 minutes and washed before continuing to other surface markers. For staining of transcription factors and Ki67 the cells were permeabilized after surface staining with FoxP3 transcription factor staining set (eBioscience) for and with Fixation/Permeabilisation Solution kit (BD Bioscience) for detection of intracellular cytokines as instructed by manufacturer. The samples were run using LSR Fortessa (BD Biosciences) and analyzed with FlowJo (BD Biosciences, LLC). The antibodies used in the study are shown in Supplementary Table X. Optimal concentration for antibodies was titrated with live PBMCs.
10
Multiparametric Flow Cytometry Analysis
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Flow cytometry analysis was performed on 1-2 x 106 freshly isolated mouse cells to immunophenotype isolated intra-hepatic and splenic regulatory and effector T cells, macrophages and monocytes. For experiments involving Foxp3-DTR mice, Cells were stained with LIVE/DEAD™ Fixable Violet Dead Cell stain kit (1:1000 dilution with PBS) (Life Technologies). Otherwise, LIVE/DEAD Fixable Green Dead cell stain was used (Life Technologies). Following Live/Dead staining, the cells were washed and resuspended in 100 µl surface staining mastermix (Table S1
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