Fresh healthy donor PBMCs were depleted of naïve CD45RA+ T cells using CD45RA MACS beads (130–045-901, Miltenyi Biotech, Germany)(22 ) to isolate memory T cells for EBVST or CMVST generation. Autologous mature DCs were pulsed at 37°C for 1 hour with 1 ng/mL EBV pepmixes (JPT Peptide Technologies, Berlin, Germany) comprising 15-mer amino acid peptides that overlapped by 11 amino acids and covered the entire protein sequence of IE-1 and pp65 for the CMVST controls and the EBV latent antigens EBNA1, LMP1, and LMP2 for the EBVSTs (16 (link)). Memory T cells were then plated with the EBV or CMV pepmix-pulsed DCs at a PBMC:DC ratio of 20:1 in a 24-well tissue culture plate. The stimulated memory T cells were fed with 10 ng/mL of IL7 and IL15, transduced with the retroviral vector on day 3, and supplemented with cytokines every 2–3 days throughout the culture. A second stimulation with pepmix-pulsed autologous irradiated PBMCs at a ratio of 1:1 was performed between days 9 and 11. A third stimulation with pepmix-pulsed irradiated PBMCs occurred 7–9 days following the second stimulation. Functional assays were performed on the days of each restimulation and 7 days after the third stimulation.
Cd45ra macs beads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD45RA MACS beads are a magnetic bead-based product used for the isolation and separation of CD45RA-positive cells from a heterogeneous cell population. The beads are coated with antibodies specific to the CD45RA antigen expressed on certain immune cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using cd45ra macs beads
1
Generating EBV-specific and CMV-specific T cells
2
Generating EBV- and CMV-Specific T Cells
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Fresh healthy donor PBMCs were depleted of naïve CD45RA+ T cells using CD45RA MACS beads (130-045-901, Miltenyi Biotec; ref. 23 ) to isolate memory T cells for EBVST or cytomegalovirus-specific T cell (CMVST) generation. Autologous mature DCs were pulsed at 37°C for 1 hour with 1 ng/mL EBV pepmixes (JPT Peptide Technologies) comprising 15-mer amino acid peptides that overlapped by 11 amino acids and covered the entire protein sequence of IE-1 and pp65 for the CMVST controls and the EBV latent antigens EBNA1, LMP1, and LMP2 for the EBVSTs (17 (link)). Memory T cells were then plated with the EBV or CMV pepmix-pulsed DCs at a PBMC:DC ratio of 20:1 in a 24-well tissue culture plate. The stimulated memory T cells were fed with 10 ng/mL of IL7 and IL15, transduced with the retroviral vector on day 3, and supplemented with cytokines every 2 to 3 days throughout the culture. A second stimulation with pepmix-pulsed autologous irradiated PBMCs at a ratio of 1:1 was performed between days 9 and 11. A third stimulation with pepmix-pulsed irradiated PBMCs occurred 7 to 9 days following the second stimulation. Functional assays were performed on the days of each resti-mulation and 7 days after the third stimulation.
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