T cell activation beads were removed and cells were washed with PBS containing 1% FBS and 2 mM EDTA. For evaluation of isolated CD4+ and CD8+ T cells, cells were blocked using normal rat serum for 15 min at room temperature and stained with anti-CD3-FITC (clone OKT3; eBioscience) and anti-CD4-PE (clone GK1.5; eBioscience) or anti-CD8-PE/Cy5 (clone 53–6.7; Biolegend) for 1 h to assess purity. Cells were fixed and permeabilized using the FoxP3 fixation and permeabilization kit from eBioscience according to the manufacturer's instructions. Subsequently, cells were incubated with the following anti-mouse antibodies in different combinations: FoxP3 (clone FJK-16s), Tbet (clone 4B10), GATA3 (clone TWAJ), RORγ (clone AFKJS-9), or isotype controls (all from eBioscience) for 1 h at room temperature. After washing, cells were analyzed by flow cytometry on a Guava EasyCyte Plus (Millipore) and analyzed using gauvaSoft Incyte software.
Anti cd4 pe clone gk1.5
Manufactured by Thermo Fisher Scientific
Anti-CD4-PE (clone GK1.5) is a fluorescently-labeled monoclonal antibody that binds to the CD4 cell surface protein. It is designed for use in flow cytometry applications.
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Lab products found in correlation
2 protocols using anti cd4 pe clone gk1.5
1
Immunophenotyping of T Cell Subsets
2
Isolation and Phenotyping of T Cells
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300 μl of blood were collected in EDTA-coated tubes. Blood samples were incubated with 3 ml of red blood cell lysis buffer (NH4Cl 150 mM, NaHCO3 10 mM and EDTA 1 mM, pH=7.4) for 10 min on ice.
After erythrocyte lysis, blood suspension was centrifuged at 1200 rpm for 5 min at 4°C. The supernatant was removed and the lysis procedure was repeated for 5 min. After centrifugation, the pellet was resuspended in 100 μl PBS + 10% fetal bovine serum (FBS, Fisher Bioblock) for cell staining.
Leukocytes were stained with anti-CD3-FITC (clone 145-2C11, BioLegend), anti-CD4-PE (clone GK 1.5, eBioscience) and anti-CD8-Alexa Fluor 700 (clone 53-6.7, BioLegend) for 30 min on ice. After washing, DAPI was added to label dead cells. Samples were analyzed using a MoFlo XDP flow cytometer (Beckman Coulter) and Summit software 5.2 version. CD4 + and CD8 + T cells were reported as percentages of CD3 + lymphocytes after excluding dead cells and cellular debris based on DAPI staining and forward and side scatter profiles.
After erythrocyte lysis, blood suspension was centrifuged at 1200 rpm for 5 min at 4°C. The supernatant was removed and the lysis procedure was repeated for 5 min. After centrifugation, the pellet was resuspended in 100 μl PBS + 10% fetal bovine serum (FBS, Fisher Bioblock) for cell staining.
Leukocytes were stained with anti-CD3-FITC (clone 145-2C11, BioLegend), anti-CD4-PE (clone GK 1.5, eBioscience) and anti-CD8-Alexa Fluor 700 (clone 53-6.7, BioLegend) for 30 min on ice. After washing, DAPI was added to label dead cells. Samples were analyzed using a MoFlo XDP flow cytometer (Beckman Coulter) and Summit software 5.2 version. CD4 + and CD8 + T cells were reported as percentages of CD3 + lymphocytes after excluding dead cells and cellular debris based on DAPI staining and forward and side scatter profiles.
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