The following antibodies, which were all purchased from eBioscience, San Diego, were used for flow cytometry: anti-mouse CD3 eFluor 450 (cat. 48-0032), anti-mouse CD4 APC-eFluor 780 (cat. 47-0042), anti-mouse CD8 APC (cat. 11-0081), anti-mouse F4/80 PE-Cy7 (cat. 25-4801), anti-mouse CD11b APC-Cy7, anti-mouse-IFN-γ PE (cat. 12-7311), anti-mouse PD-1 PE (12-9985), anti-mouse PD-L1 PE (cat. 12-5982), anti-mouse IL-10 (cat. 11-7101) and anti-mouse Ki67 eFluor 450 (cat. 48-5698). Two million cells per sample were used for flow cytometry. Cells were incubated with Fc Block for 15 minutes at 4°C in darkness to block the FcγR and then stained with fluorochrome-conjugated antibodies for 30 minutes at 4oC also in darkness. Cells were then analyzed using a Canto II (BD Bioscience, CA) 8-color flow cytometer, and data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
Anti mouse cd3 efluor450
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse CD3-eFluor450 is a monoclonal antibody that binds to the CD3 complex on the surface of mouse T cells. It is conjugated to the eFluor450 fluorescent dye, which can be detected using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Beckman gallios flow cytometer, by Beckman Coulter (2 mentions)
Canto 2, by BD (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Flow cytometry, by BD (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Anti mouse cd25 apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 pe, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti mouse cd3 efluor450
1
Multicolor Flow Cytometry for Immune Cell Profiling
2
Cytokine Profiling of Mediastinal Lymph Nodes
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Mediastinal lymph nodes (MLN) were collected and single cell suspension was prepared. The cells were re-suspended in DMEM and incubated with a freshly prepared cocktail containing 50 ng/ml PMA, 500 ng/ml ionomycin and 10 μg/ml brefeldin A (Sigma-Aldrich, Oakville, ON) for 4h at 37°C and 5% CO2. Extracellular staining was performed using anti-mouse CD3 e-Fluor® 450 (Clone: 17A2) and CD4-FITC (Clone: RM4-5) both from eBioscience. After fixation with paraformaldehyde for 15 min at 4°C, cells were permeabilized with 0.1% saponin. Finally, intracellular staining was performed using anti-mouse IL-4-PE (Clone: 11B11), IL-17A-PE (Clone: eBio17B7), and IFN-γ-PE (Clone: XMG1.2). Samples were acquired on a FACSCantoII and analyzed using FlowJo software.
3
Apoptosis Detection by Flow Cytometry
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The apoptosis rate was detected by flow cytometry (Becton Dickinson, San Jose, CA, USA) using Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGEN Biotech, Jiangsu, China) according to previous research (17 (link)). Annexin V-FITC (−)/Propidium Iodide (PI) (−) (the lower left) are normal cells, Annexin V-FITC (+)/PI (−) cells (the lower right) are early apoptotic cells, Annexin V-FITC (+)/PI (+) (the upper right) are late apoptosis cells, and Annexin V (−)/PI (+) (the upper left) are necrotic cells. For immune cells, anti-mouse CD3-eFluor450 (eBioscience, CA, USA) was added to the peripheral blood and incubated for 30 min at room temperature in the dark. Then, the erythrocyte lysis solution was added to the sample and incubated for another 20 min. The cells were washed and re-suspended with PBS, and then analyzed by Beckman Gallios flow cytometer (Beckman Coulter, Inc., CA, USA).
4
Flow Cytometric Analysis of Mouse Immune Cells
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A total of 2.5 μL of anti-mouse CD3-eFluor450, 0.625 μL of anti-mouse CD4-PE, 0.625 μL of anti-mouse CD25-APC (all from eBioscience, California, USA) were added to 100 μL of peripheral blood. Following incubation at room temperature in the dark for 15–30 min, 1 mL of erythrocyte lysis solution was added to the samples and incubated for 15–20 min. The cells were washed with 2 mL phosphate-buffered saline (PBS) and resuspended in 500 μL PBS. The cells were then analyzed using a Beckman Gallios flow cytometer (Beckman Coulter, Inc., California, USA).
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