Author: Banu, Nasirah; Chia, Adeline; Ho, Zi Zong; Garcia, Alfonso Tan; Paravasivam, Komathi; Grotenbreg, Gijsbert M.; Bertoletti, Antonio; Gehring, Adam J.
Title: Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections Document date: 2014_2_25
ID: 44w6omdp_31
Snippet: PBMC were stimulated with 600 U/ml interleukin-2 (IL-2; R&D Systems) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, CA) for 48 h. Untreated 24 well tissue culture plates were coated with 30 ug/ml Retronectin (Takara Bio) overnight at 4uC one day prior to transduction. Wells were then washed with HBSS and blocked with PBS 2% BSA. Lymphocytes were harvested, washed, counted and 5 3 10 5 cells plated into retronectin coated wells and mixed wi.....
Document: PBMC were stimulated with 600 U/ml interleukin-2 (IL-2; R&D Systems) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, CA) for 48 h. Untreated 24 well tissue culture plates were coated with 30 ug/ml Retronectin (Takara Bio) overnight at 4uC one day prior to transduction. Wells were then washed with HBSS and blocked with PBS 2% BSA. Lymphocytes were harvested, washed, counted and 5 3 10 5 cells plated into retronectin coated wells and mixed with 1.5-2 ml of unconcentrated retroviral supernatants collected as described above. After 24 h, the viral supernatant was replaced with Aim-V medium with 2% human AB serum, supplemented with 100 U/ml IL-2. TCR surface expression was tested with HLA-pentamers (ProImmune), HLA-tetramer produced by peptide exchange 39 or anti-Vb antibodies 72 h post transduction. Functionality of TCR redirected T cells was tested 7 days post transduction as described below.
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