The lymphocytes were washed with cold PBS containing 0.1% NaN3 and 1% FBS. A total of 106 cells were incubated with APC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD4 and FITC-conjugated anti-mouse CD8 (BD Biosciences) for 20 min at room temperature. The cells were washed in PBS containing 0.1% FBS and analyzed by flow cytometry (BD Calibur™).
Apc conjugated anti mouse cd3
Manufactured by BD
Sourced in United States
APC-conjugated anti-mouse CD3 is a monoclonal antibody that binds to the CD3 antigen expressed on the surface of mouse T cells. The antibody is conjugated with allophycocyanin (APC), a fluorescent dye, which allows for the detection and identification of T cells in flow cytometry applications.
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Lab products found in correlation
Facscalibur, by BD (2 mentions)
Buv395 conjugated anti mouse cd8b antibodies, by BD (2 mentions)
Fitc conjugated anti mouse cd8, by BD (1 mentions)
Pe conjugated anti mouse cd4, by BD (1 mentions)
Fitc conjugated anti mouse igg, by BD (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
Fitc conjugated anti mouse cd11b, by BD (1 mentions)
Apc conjugated anti mouse gr 1, by BD (1 mentions)
Pecy7 conjugated anti mouse nk1, by BD (1 mentions)
Anti mouse cd16 cd32 fc block, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
Fitc conjugated anti mouse cd19, by BD (1 mentions)
Pe conjugated anti mouse f4 80, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Pe conjugated anti mouse pd 1, by BD (1 mentions)
Anti cd20 antibody, by BioLegend (1 mentions)
Sa271g2, by BioLegend (1 mentions)
Anti cd8 antibody, by BioXCell (1 mentions)
6 protocols using apc conjugated anti mouse cd3
1
Flow Cytometry Analysis of T Cell Subsets
2
Alloantibody Levels Post-Transplant
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The circulating anti-donor IgG and IgM in the recipients’ sera on day 7 posttransplant were evaluated by flow cytometry. In brief, BALB/c mouse splenocytes were isolated and incubated at 4°C for 30 min with sera (1:40 dilutions) from all experimental groups. The cells were washed and then incubated with APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse IgG, and PE-conjugated anti-mouse IgM (BD Biosciences, USA). The stained cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences). Data are expressed as geometric mean fluorescence intensity (GMean), which represents the intensity of antibody binding. Naive sera from C57BL/6 mice were used as negative controls.
3
CD8+ T Cell Depletion Protocol
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Optimization experiments were performed to determine the appropriate dosages required to achieve CD8+ T cell depletions. A129 mice were administered intraperitoneally (i.p.) with anti-CD8a antibody (Bio X Cell #BE0061). A second antibody injection at the same doses was given 2 days later, as previously described [20] (link). Flow cytometry was performed one day after the second injection on submandibular blood stained with APC-conjugated anti-mouse CD3 (BD Cat#565643) and BUV395-conjugated anti-mouse CD8b antibodies (BD Cat#740278) to evaluate the optimal dose for successful CD8+ T cell depletion.
For CD8 T-cell depletion, 10μg of anti-CD8a antibody diluted in 200μL PBS was injected i.p. at one and three days before challenge with H/PF/2013. Successful depletion of CD8+ cells was confirmed via flow cytometry.
For CD8 T-cell depletion, 10μg of anti-CD8a antibody diluted in 200μL PBS was injected i.p. at one and three days before challenge with H/PF/2013. Successful depletion of CD8+ cells was confirmed via flow cytometry.
4
Intracellular PTX3 and Immune Cell Analysis
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To analyze PTX3 intracellular expression, transfected HEK 293T cells were fixed with 2% PFA and permeabilized with 0.1% Saponin (Sigma Aldrich) in PBS. Indirect intracellular staining was performed with rat anti-PTX3 (MNB4, Abcam) primary antibody, followed by AF488-conjugated anti-rat (Life Technologies) secondary antibody. To identify the various cell populations present in splenocytes, peritoneal lavage and quadriceps harvested from mice, cells were first incubated with anti-mouse CD16 / CD32 (FC block, BD Pharmingen) and stained with the following antibodies: APC-conjugated anti-mouse GR1, PE-conjugated anti-mouse F4/80, FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse Ly6c, APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD19, PE-conjugated anti-mouse CD45, or PE-Cy7-conjugated anti-mouse NK1.1 (BD Pharmingen). For detection of alphavirus antigens, indirect intracellular staining was performed using mouse monoclonal anti-alphavirus (3581, Santa Cruz) primary antibody, followed by AF488-conjugated anti-mouse (Life Technologies) secondary antibody. Data acquisition was performed using CyanADP (Beckman Coulter), and analysis was done by Kaluza Flow Analysis Software (Beckman Coulter).
5
Multiparametric Flow Cytometry for Immune Profiling
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For flow cytometry, allophycocyanin (APC)-conjugated anti-mouse CD3 (BD Biosciences, Palo Alto, CA), phycoerythrin (PE)-conjugated anti-mouse PD-1, fluorescein isothiocyanate (FITC)-conjugated CD127, and PE-Cy5.5-conjugated anti-mouse CD4 or CD8 (BD Biosciences Pharmingen) monoclonal (m) Abs were used for flow cytometry. For the flow cytometric analysis, 105 cells were labeled in a fluorescence-activated cell sorter (FACS) buffer (PBS/2% FCS/0.1% sodium azide), fixed in 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO), and analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA).
6
Depleting B and T Cells in SARS-CoV-2 Challenge
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All CD20+ B and CD8+ T cell depletion studies followed by wild-type SARS-CoV-2 challenge were carried out by adapting a previously published approach. B cells were depleted from hACE2 transgenic mice through the intraperitoneal (i.p.) administration of 50 μg of anti-CD20 antibody (BioLegend SA271G2) at 72 h (−3 days) and 24 h (−1 day) before vaccination with LUNAR-COV19. For CD8+ T cell depletion, 5 μg of anti-CD8+ antibody (Bio X Cell, Cat#BE0061) was injected i.p. at −3 days and −1 day before challenge with SARS-CoV-2. For control mice, isotype IgG2b mouse control antibody (Bio X Cell, Cat#BE0090) was similarly injected. Successful depletions of CD20+ B and/or CD8+ T cells were confirmed via flow cytometry. Briefly, 24 h after the second injection of anti-CD20+ and/or anti-CD8+ antibodies, sub-mandibular blood was stained with AF488-conjugated anti-mouse CD45R (BD, Cat#557669), APC-conjugated anti-mouse CD3 (BD, Cat#565643), and BUV395-conjugated anti-mouse CD8b antibodies (BD, Cat#74027).
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