Dye-labeled free OMVs or OM-NDs were administered into mice via the hock at a protein dose of 20 μg. After 6 h, the mice were euthanized, and the draining popliteal lymph nodes were collected. The fluorescence in each lymph node was visualized and quantified using an IVIS Lumina in vivo imaging system. To analyze the cellular-level distribution, each draining popliteal lymph node was mechanically sheared and extruded through a 40-μm cell strainer (Bel-Art) to form a single-cell suspension. Then, 5 × 105 cells were blocked with TruStain FcX PLUS anti-mouse CD16/32 at 4 °C for 10 min, followed by staining with LIVE/DEAD fixable aqua, PE/Cy7 anti-mouse CD11c (N418, BioLegend), FITC anti-mouse CD3 (17A2, BioLegend), Pacific Blue anti-mouse CD19 (6D5, BioLegend), APC/Cy7 anti-mouse CD11b (M1/70, BioLegend), PE anti-mouse F4/80 (BM8, BioLegend), and PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) (RB6-8C5, BioLegend) for 30 min. Finally, the cells were washed and resuspended in PBS containing EDTA and BSA. Data was collected on a Becton Dickinson LSRFortessa flow cytometer, and analysis was performed using FlowJo software.
Fitc anti mouse cd3 17a2
Manufactured by BioLegend
The FITC anti-mouse CD3 (17A2) is a monoclonal antibody that recognizes the CD3 complex on mouse T cells. CD3 is a key component of the T cell receptor complex and is expressed on all mature T cells. The antibody is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate), allowing for the detection and analysis of mouse T cells by flow cytometry.
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Lab products found in correlation
Pacific blue anti mouse cd19 6d5, by BioLegend (2 mentions)
Pe anti mouse f4 80 bm8, by BioLegend (2 mentions)
Percp anti mouse ly 6g ly 6c gr 1 rb6 8c5, by BioLegend (2 mentions)
Lsrfortessa flow cytometer, by FlowJo (1 mentions)
Trustain fcx plus anti mouse cd16 32, by BioLegend (1 mentions)
Pe cyanine7 anti mouse cd11c n418, by BioLegend (1 mentions)
Xenogen ivis 200 imaging system, by PerkinElmer (1 mentions)
Lsrii flow cytometer, by FlowJo (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using fitc anti mouse cd3 17a2
1
Tracking OMVs in Lymph Nodes
2
Lymph Node Trafficking of DiD-labeled α7-NTs
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CD1 mice were subcutaneously injected in their hock regions with DiD-labeled α7-NTs, and their draining lymph nodes were collected at 6 or 24 h after injection for imaging and quantification using a PerkinElmer Xenogen IVIS 200 imaging system. For cellular level analysis, single-cell suspensions were generated from lymph node samples by manual dissociation, passage through Flowmi 40-μm cell strainers (Bel-Art), and 2 washes with PBS. The cell suspensions were then incubated with a LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen), blocked with 1 % BSA in PBS, and stained with a panel of antibodies, including FITC anti-mouse CD3 (17A2, BioLegend), Pacific Blue anti-mouse CD19 (6D5, BioLegend), PE/cyanine7 anti-mouse CD11c (N418, BioLegend), PE anti-mouse F4/80 (BM8, BioLegend), APC/cyanine7 anti-mouse CD11b (M1/70, BioLegend), and PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) (RB6-8C5, BioLegend). B cells were defined as CD19+, macrophages were defined as CD11b+F4/80+, dendritic cells were defined as CD11c+F4/80-, granulocytes were defined as CD11b+Gr-1+, and T cells were defined as CD3+. Unstained, single-stained controls, and fluorescence-minus-one controls were used for compensation and gating purposes. Data were acquired using a Becton Dickinson LSR II flow cytometer and analyzed using FlowJo v10.4 software.
3
Mouse Lymphocyte Surface Staining
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PBMCs were obtained from patients with epilepsy at Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine. This project was approved by the local ethics committee (SCMCIRB— K2019026-2). Informed consent was obtained from each patient, and the study protocol was approved by the Clinical Research Ethics Committee of Shanghai Children’s Medical Center and complied with all relevant ethical regulations.
For mouse lymphocyte surface staining, single-cell suspensions were stained with fluorophore-conjugated anti-mouse antibodies diluted in 50 μL PBS containing 2% FBS on ice for 30 min in the dark. Cells were washed once with 1× PBS containing 2% FBS and re-suspended in 1× PBS containing 2% FBS for flow cytometry or cell sorting. Normally, Fixable Viability Stain 700 (AF700, BD) or L/D PE-CF594 (BD), FITC anti-mouse CD3 (17A2, Biolegend), Brilliant Violet 421™ anti-mouse CD4 (GK1.5, Biolegend), Brilliant Violet 421™ anti-mouse CD8a (53-6.7, Biolegend), APC-eFluor 780 anti-mouse CD4 (RM4-5, eBioscience), APC-eFluor 780 anti-mouse CD8 (53-6.7, eBioscience) and PE anti-mouse B220 (RA3-6B2, eBioscience) and Alexa Fluor® 647 anti-mouse CD19 (6D5, Biolegend) were used for staining.
For mouse lymphocyte surface staining, single-cell suspensions were stained with fluorophore-conjugated anti-mouse antibodies diluted in 50 μL PBS containing 2% FBS on ice for 30 min in the dark. Cells were washed once with 1× PBS containing 2% FBS and re-suspended in 1× PBS containing 2% FBS for flow cytometry or cell sorting. Normally, Fixable Viability Stain 700 (AF700, BD) or L/D PE-CF594 (BD), FITC anti-mouse CD3 (17A2, Biolegend), Brilliant Violet 421™ anti-mouse CD4 (GK1.5, Biolegend), Brilliant Violet 421™ anti-mouse CD8a (53-6.7, Biolegend), APC-eFluor 780 anti-mouse CD4 (RM4-5, eBioscience), APC-eFluor 780 anti-mouse CD8 (53-6.7, eBioscience) and PE anti-mouse B220 (RA3-6B2, eBioscience) and Alexa Fluor® 647 anti-mouse CD19 (6D5, Biolegend) were used for staining.
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