Splenocytes were labeled with cell proliferation dye eF450 (eBioscience) per the manufacturer’s instructions. CD8+ T cells were enriched using an AutoMACS (Miltenyi Biotech) and CD8α MicroBeads (Miltenyi Biotech) and i.v. injected into KbDb−/−.SJL recipients. Two hours post-injection, recipients were left naive, stimulated with 50 mg CPG ODN (Invivogen) + 100 μg Poly(I:C) (Sigma-Aldrich), or infected with MCMV. On day 2, mice received second dose of 50 μg CPG ODN + 100 μg Poly(I:C). Donor CD8+ T cell proliferation was analyzed on day 4.
Ef450
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EF450 is a compact centrifuge designed for general-purpose laboratory applications. It offers a maximum speed of 4,500 rpm and accommodates a variety of sample tube sizes. The centrifuge provides consistent and reproducible results for a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Cpg odn, by InvivoGen (2 mentions)
Automacs, by Miltenyi Biotec (2 mentions)
Poly 1 c, by Merck Group (2 mentions)
Cd8α microbeads, by Miltenyi Biotec (2 mentions)
Percp cy5, by Thermo Fisher Scientific (2 mentions)
Pdgfrα cd140a, by Thermo Fisher Scientific (2 mentions)
Streptavidin qdot605, by Thermo Fisher Scientific (2 mentions)
Lsrii fortessa, by BD (2 mentions)
Celesta flow cytometer, by BD (2 mentions)
Cell trace yellow proliferation dye, by Thermo Fisher Scientific (2 mentions)
Cd16 32 pe, by Thermo Fisher Scientific (1 mentions)
Sca 1 pe cy7, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Mitosox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by Tree Star (1 mentions)
Facs lsrfortessa, by BD (1 mentions)
Ef670, by Thermo Fisher Scientific (1 mentions)
Dnase, by Promega (1 mentions)
Pyronin y, by Merck Group (1 mentions)
16 protocols using ef450
1
Proliferation of Adoptive CD8+ T Cells
2
Immunophenotyping and Functional Characterization of Murine Hematopoietic Stem and Progenitor Cells
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Peripheral blood samples were stained with CD45 PerCP-Cy5.5/APC Cy7, B220 APC/PE Cy7, Gr1 Alexa Fluor 700, Mac1/CD11b, CD3e APC/PE, CD4 PE, and CD8a APC/PE.
Whole bone marrow cells were stained as above for mature lineages. Cells were also stained for Lineage– Sca1+ Kit+ CD48– CD150+ (LSK SLAM) with biotin-conjugated anti-mouse lineage antibodies (Ter119, B220, Gr1, CD11b, CD3e) followed by staining for streptavidin V500/eF450 (eBioscience [eF450]), c-Kit APC eF780/APC (eBioscience [APC eF780]), Sca1 PE Cy7, CD48 AF700/BV605 (Biolegend [AF700]) CD150 APC/PE (eBioscience [APC]; Fisher Scientific [PE]). Bone marrow cells were further stained with CD16/32 PE (eBioscience) and CD34 eF450 (eBioscience) to immunostain for committed progenitor populations. For mitochondrial function, cells immunostained for LSK SLAM were incubated at 37°C in 5% CO2 for 30 min with either MitoSOX Deep Red Reagent (1 mM, Invitrogen) or tetramethylrhodamine ester (0.1 mM, Sigma Aldrich). Caspase 1 activity was determined using the FLICA assay (Corning), according to the manufacturer’s recommendations. Samples were then analyzed using a BD LSR II, BD LSR Fortessa, or BD Canto III (BD Biosciences). All antibodies were obtained from BD Biosciences, unless otherwise noted.
Whole bone marrow cells were stained as above for mature lineages. Cells were also stained for Lineage– Sca1+ Kit+ CD48– CD150+ (LSK SLAM) with biotin-conjugated anti-mouse lineage antibodies (Ter119, B220, Gr1, CD11b, CD3e) followed by staining for streptavidin V500/eF450 (eBioscience [eF450]), c-Kit APC eF780/APC (eBioscience [APC eF780]), Sca1 PE Cy7, CD48 AF700/BV605 (Biolegend [AF700]) CD150 APC/PE (eBioscience [APC]; Fisher Scientific [PE]). Bone marrow cells were further stained with CD16/32 PE (eBioscience) and CD34 eF450 (eBioscience) to immunostain for committed progenitor populations. For mitochondrial function, cells immunostained for LSK SLAM were incubated at 37°C in 5% CO2 for 30 min with either MitoSOX Deep Red Reagent (1 mM, Invitrogen) or tetramethylrhodamine ester (0.1 mM, Sigma Aldrich). Caspase 1 activity was determined using the FLICA assay (Corning), according to the manufacturer’s recommendations. Samples were then analyzed using a BD LSR II, BD LSR Fortessa, or BD Canto III (BD Biosciences). All antibodies were obtained from BD Biosciences, unless otherwise noted.
3
Isogenic p53 Cell Lines Profiling
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Three independent isogenic p53WT/WT and p53KO/KO cell lines were prepared by trypsinization. Antibodies against murine CD45, Mac1, Gr1, F4/80, B220, IgM, CD2, CD3, CD4, CD8, Ter119, Sca1, CD51, PDGFRα (CD140a), CD31, either biotinylated or conjugated with eF450, PE, PerCP-Cy5.5, or APC were obtained from eBioscience (San Diego, CA) or BD Pharmingen. Biotinylated antibodies were detected with Streptavidin-Qdot605 (Invitrogen). Flow cytometry was performed on an LSRII Fortessa (BD Bioscience) interfaced with Cell Quest software, data were analysed on FlowJo (TreeStar).
4
Antibody-Mediated Complement-Dependent Cytotoxicity
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B16-OVA and B16-OVA-Thy1.1 cells were detached with 2 mmol/L Ethylenediamine tetraacetic acid (EDTA; Gibco) and were prestained with eF450 and eF670 (eBioscience) respectively, following manufacturers’ instructions. The stained cells were then mixed in 1:1 ratio in 96-well round bottom plate (5 × 105 cells per well). Cells were washed three times with FACS buffer (1% FBS in PBS) at 400 × g for 3 minutes at 4°C and incubated with indicated antibodies at 50 μg/mL (50 μL/well) for 30 minutes at 4°C in the dark. Next, the cells were washed three times and were incubated with prewarmed Rabbit Complement (RC; Cedarlane) diluted 1:8 in IMDM complete media (50 μL of RC/well). The cells were incubated for 1 hour at 37°C, after which DNAse (Promega; 1 U/μL) diluted in FACS buffer was added and the cells were washed three times. Finally, the cells were resuspended in 150 μL FACS buffer with 1 mg/mL propidium iodide (PI; Sigma-Aldrich). A total of 100 μL of the stained cells were analyzed on a FACS LSRFortessa (BD) using the software program BD FACSDiva. Further analysis was performed with FlowJo and shown results plotted in GraphPad.
5
Analyzing Productively Infected Cells
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To analyze productively infected cells, 2.5x105 cells were stained for CD4 (clone: S3.5, APC, BD), fixable viability dye (eF450, eBiosciences) and intracellular p24-gag (BD) and were analyzed on a Celesta flow cytometer (BD) as previously performed [34 (link)]. For proliferation studies, cells were stained for CD4, viability and intracellular Ki67 (clone: 6604665, FITC, Biolegend). To analyze cellular RNA/ DNA content in order to determine cell cycle state, cells were stained with 7-AAD (Millipore Sigma, MA) and PyroninY (Sigma, MO) as previously done [84 (link)]. To label cells prior to co-culture assays, cells were stained with Cell Trace Yellow proliferation dye (ThermoFisher, MA). Flow cytometry analysis was performed in FlowJo software (BD) and further statistical analysis was performed in GraphPad Prism (CA).
6
HIV Infection Cell Analysis Protocol
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To analyze productively infected cells, 2.5 × 105 cells were stained for CD4 (clone S3.5, APC; BD), fixable viability dye (eF450; eBiosciences), and intracellular p24-Gag (Kc57, FITC; BD) and were analyzed on a Celesta flow cytometer (BD) as previously performed, with 1.5 × 105 events collected per condition (35 (link)). To label cells prior to coculture assays, cells were stained with Cell Trace Yellow proliferation dye (ThermoFisher, MA) according to the manufacturer’s instructions. To determine percent expression of total and phosphorylated SAMHD1, CD4 T cells were stained with pSAMHD1 (Cell Signaling Technologies) and total SAMHD1 (Origene) according to manufacturer protocols. For determination of CD4 and coreceptor MFI, cells were stained for CD4 (clone S3.5, APC; BD), fixable viability dye (eF450; eBiosciences), CXCR4 (PE-Cy7; eBiosciences), and CCR5 (BV786; BioLegend) and were analyzed on a Celesta flow cytometer (BD) as previously performed (35 (link)).
7
MSC Immunomodulation via IDO Expression
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MSC were seeded in a 6-well plate at a density of 5000 cells/cm2 in RPMI. Cells were allowed to attach and were then treated for 72 h either with IFNγ (10 ng/mL), Tryp (100 µg/mL) or Epac (1 µM). After trypsinisation and washing, MSC were resuspended in PBS, stained with fixable viability dye eF450 (1:4000 final dilution; eBioscience, Frankfurt am Main, Germany) for 30 min at 4 °C. After washing once, cell suspension was incubated with intracellular fixation buffer (eBioscience, Frankfurt am Main, Germany) during 30 min at room temperature (RT). After washing/centrifuging samples twice with 1× permeabilisation buffer (eBioscience, Frankfurt am Main, Germany), cells were stained with IDO-PE (eBioscience, Frankfurt am Main, Germany) in 1× permeabilisation buffer for 30 min. Cells were washed and analysed immediately at BD FACS Canto II (BD Biociences, Heidelberg, Germany). MSC without IFNγ stimulation served as control. Data are represented as MFI after subtraction of MFI’s respective control.
8
Multicolor Flow Cytometry Panel
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The expression of CD31 (AF488, #MCA1334A488, BioRad, Oxford, UK), CD45 (PE, #MCA43PE, BioRad), CD73 (eF450, #48-0731-82, eBioscience Inc., San Diego, CA) and CD90 (PE-Cy5, #ab95809, Abcam, Cambridge, UK) was analyzed by polychromatic flow cytometry (LSRII; Becton Dickinson, Franklin Lakes, NJ) with fluorochrome-conjugated monoclonal antibodies. Cells at passage 2 were detached by 4% lidocaine (Sigma, St Louis, MO). BD CompBeads particles (BD Biosciences) were used to calculate the compensation of fluorescence spillover. Only events from alive cells in both morphogate and singulet gates were analyzed.
9
Murine Immune Cell Immunophenotyping
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OS cell lines (less than passage 5 from establishment) were prepared by trypsinization. Antibodies against murine CD45, Mac1, Gr1, F4/80, B220, IgM, CD2, CD3, CD4, CD8, Ter119, Sca1, CD51, PDGFRα (CD140a), CD31, either biotinylated or conjugated with eF450, PE, PerCP-Cy5.5, or APC were obtained from eBioscience (San Diego, CA) or Pharmingen. Biotinylated antibodies were detected with Streptavidin-Qdot605 (Invitrogen). Flow cytometry was performed on an LSRII Fortessa (BD Bioscience) interfaced with CellQuest software, data was analyzed on FlowJo (TreeStar).
10
Proliferation of Adoptive CD8+ T Cells
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