Resting CD8+ T cells were isolated from Ficoll-purified PBMC isolated from leukapheresis products (Memorial Blood Center, St. Paul, MN) in a two-step process by first depleting CD25+ cells using using (cGMP)-grade anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) on an AutoMACS (Miltenyi Biotec). CD8+ cells were then purified from the CD25− fraction using negative selection (CD8+ T cell isolation kit, Miltenyi Biotec). Purified cells were stimulated for 72 h with αCD3/αCD28 beads (InVitrogen) + IL-2 (300U/ml). After 20 h, A-438079 (25µM) or PBS was added to the cultures. Cell numbers were analyzed by Neubauer chamber counting. Survival was analyzed by Trypan blue staining during microscope counting and confirmed by Live-Dead (Tonbo Biosciences) staining using flow cytometry.
Ficoll purified pbmc
Manufactured by Miltenyi Biotec
Sourced in Sao Tome and Principe
Ficoll-purified PBMC is a laboratory reagent used to isolate peripheral blood mononuclear cells (PBMCs) from whole blood samples. It employs a density gradient centrifugation method to separate the mononuclear cell fraction, which includes lymphocytes and monocytes, from other blood components.
Automatically generated - may contain errors
2 protocols using ficoll purified pbmc
1
Isolation and Stimulation of Resting CD8+ T Cells
2
Isolation and Stimulation of Resting CD8+ T Cells
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Resting CD8+ T cells were isolated from Ficoll-purified PBMC isolated from leukapheresis products (Memorial Blood Center, St. Paul, MN) in a two-step process by first depleting CD25+ cells using using (cGMP)-grade anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) on an AutoMACS (Miltenyi Biotec). CD8+ cells were then purified from the CD25− fraction using negative selection (CD8+ T cell isolation kit, Miltenyi Biotec). Purified cells were stimulated for 72 h with αCD3/αCD28 beads (InVitrogen) + IL-2 (300U/ml). After 20 h, A-438079 (25µM) or PBS was added to the cultures. Cell numbers were analyzed by Neubauer chamber counting. Survival was analyzed by Trypan blue staining during microscope counting and confirmed by Live-Dead (Tonbo Biosciences) staining using flow cytometry.
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