Isolation of mouse liver non-parenchymal cells was as described in this section. Subsequently, antibodies including anti-F4/80-APC (17-4801-82), anti-CD11c-APC (17-0114-82), anti-CD3e-APC (17-0031-82), and anti-CD31-APC (17-0311-82, eBioscience, San Diego, CA) were added to the cell suspension, respectively. After 30 min of incubation at 4°C in the dark, the cells were washed with PBS and incubated with fixation/permeabilization buffer (eBioscience, San Diego, CA). Then, the cells were washed and stained with anti-NLRP3/NALP3-750 (IC7578S, R&D Systems, Eugene, Oregon) for 60 min at 4°C and analyzed with a flow cytometer. FACS was performed on a FACSAria and analyzed with FACSDiva 4.1 (BD Biosciences).
Anti cd3e apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD3e-APC is a mouse monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It is conjugated with the fluorescent dye APC (Allophycocyanin).
Automatically generated - may contain errors
Lab products found in correlation
Facs diva 4, by BD (1 mentions)
Anti f4 80 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd11c apc, by Thermo Fisher Scientific (1 mentions)
Anti cd31 apc, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Oct medium, by Thermo Fisher Scientific (1 mentions)
Rabbit anti claudin 1, by Thermo Fisher Scientific (1 mentions)
Anti cd45 apc cyanine7, by BioLegend (1 mentions)
Anti iga fitc, by BD (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti il 17a pe, by Thermo Fisher Scientific (1 mentions)
Stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti cd3e apc
1
Isolation and Analysis of Mouse Liver Non-Parenchymal Cells
2
Immunohistochemical Analysis of Gut and Kidney Tissues
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Kidneys were embedded in OCT medium (Fisher Scientific) and snap-frozen at − 80 °C. Ileum and colon were prepared as “Swiss rolls” before being embedded in OCT medium and snap-frozen. 7 µm thick sections were mounted on histology slides set at room temperature for 20 min before being placed into PBS to dissolve the OCT. The sections were then fixed with cold acetone for 10 min, washed 3 times, and blocked with 10% normal rat serum (Equitech-Bio) in PBS for 30 min. Renal tissue sections were stained with anti-CD3e-APC (1:50; eBioscience 145-2C11). The ileum sections were stained with rabbit anti-Claudin-1 (1:100 dilution; Invitrogen MH25) followed by goat anti-rabbit IgG-AF700 (1:2000; Invitrogen A-21038). The colon sections were stained with anti-CD45-APC/Cyanine7 (1:25 dilution; Biolegend 30-F11) and anti-IgA-FITC (1:50 dilution; BD Biosciences). Fluorescence intensity was analyzed using ImageJ.
3
Lymphocyte Isolation and Th17 Characterization
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The lymphocytes in the thymus, spleen, and peripheral blood were extracted with lymphocyte separation medium and washed with PBS. The obtained mononuclear cells (1 × 106/mL) were incubated with 2 μL of stimulation cocktail (eBioscience, San Diego, CA, USA) in RPMI 1640 with 10% fetal calf serum at 5% CO2 and 37°C for 5 hours. After centrifuging and resuspending with PBS, the cells were stained with surface-specific Abs (anti-CD3e-APC and anti-CD4-FITC, all purchased from eBioscience (San Diego, CA, USA)) at 4°C in the dark for 30 min. After centrifuging and resuspending with PBS, the suspension was incubated with fixation/permeabilization solution (BD Biosciences, Sparks, MD, USA) and stained with anti-IL-17A-PE (eBioscience, San Diego, CA, USA) at 4°C in the dark for 30 min. Acquisition was done on a FACSCalibur flow cytometer (BD Biosciences, Sparks, MD, USA), and FlowJo software was used for analysis.
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