In vitro expansion of epitope-specific CD8+ T-cell lines was performed as described previously by our group87 (link). Briefly, freshly thawed cryopreserved autologous PBMCs were plated in a 48 well plate at 1.2 × 106 cells/ml in serum free RPMI media. Supernatants containing non-adherent cells were removed after a two-hour incubation at 37°C. Adherent cells, mainly monocytes, were irradiated (3,300 rad, 45 min) and pulsed with the appropriate autologous peptide (10 μM) for 2 hrs. CD8+ T cells were isolated from the non-adherent cells using the CD8+ untouched isolation kit (MACS Miltenyi Biotec) and were plated onto peptide-pulsed monocytes in the presence of complete media (RPMI+10% Hyclone serum) containing IL-7 (25 ng/ml). The CD8+ T-cell culture was maintained by adding IL-2 (50 U/ml) every 2 to 3 days and re-stimulating the CD8+ T cells with peptide-pulsed monocytes as described above on day 7. On day 13, T-cell lines were tested for cytokine responses to the cognate HIV peptide in a 6 hr ICS assay as outlined above.
Cd8 untouched isolation kit
Manufactured by Miltenyi Biotec
The CD8+ untouched isolation kit is a tool designed for the isolation of untouched CD8+ T cells from various samples, such as peripheral blood mononuclear cells (PBMCs). The kit utilizes a magnetic bead-based negative selection approach to isolate the target cells without directly labeling them, preserving their native properties.
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3 protocols using cd8 untouched isolation kit
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Expansion of Epitope-Specific CD8+ T Cells
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Expansion of Epitope-Specific CD8 T Cells
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Epitope-specific CD8 T-cell lines were expanded in vitro as previously described [14 (link)]. Briefly, cryopreserved PBMCs (obtained from chronically HIV-1 infected patients) were thawed and plated in a 48-well plate at 1.2×106 cells/ml in serum free RPMI media. Plates were incubated at 37C/5% CO2 for two hours, after which media containing non-adherent cells was removed. Adherent cells were irradiated at 3,000 rad and pulsed with the appropriate peptide at 10 μM for 2 h. Autologous CD8 T cells were isolated from the same PBMC sample using the CD8 untouched isolation kit (MACS Miltenyi Biotec). CD8 T cells were then plated at 0.5×106 cells/well onto the peptide-pulsed monocytes in the presence of complete media (RPMI+10% FBS) containing IL-7 (25 ng/ml). IL-2 (50U/ml) was added to the culture on the second day. The culture was then maintained by replacing half the media with freshly made media containing IL-2 (50U/ml) every three days, and CD8 T cells were re-stimulated on day seven (and weekly thereafter) with peptide-pulsed monocytes. CD8 T cell clone (SL9) was a gift from Dr. June Kan-Mitchell.
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Expansion of Epitope-Specific CD8+ T Cells
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