Lentiviral vectors to express SABRs or TCRs were packaged using previously described procedures19 (link). Briefly, 5×106 HEK-293T cells were plated on poly-L-Lysine coated plates for 24 hours, followed by transfection of a mixture of the lentiviral shuttle plasmid, pMDG-VSVG, and pCMV-RD8.9 (a kind gift from Donald B. Kohn) using TransIT-293 (Mirus Bio) and OPTI-MEM (Life Technologies). Viruses were filtered through 0.45 micron syringe filters (Milipore) and stored at −80°C until further use. To transduce Jurkat, NFAT-GFP-Jurkat, or Primary T cells, 2-5×105 cells were plated in culture medium and mixed with an equal volume of thawed virus in 12-well plates for three days. For NFAT-GFP-Jurkat cells, G-418 was added to the transduction mixture 48 hours later.
0.45 micron syringe filters
Manufactured by Merck Group
The 0.45 micron syringe filters are a type of lab equipment used for filtration. They are designed to remove particulates and contaminants from liquids by passing them through a 0.45 micron pore size membrane.
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2 protocols using 0.45 micron syringe filters
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Lentiviral Transduction of T Cells
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Lentiviral Transduction of T Cells
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Lentiviral vectors to express SABRs or TCRs were packaged using previously described procedures19 (link). Briefly, 5×106 HEK-293T cells were plated on poly-L-Lysine coated plates for 24 hours, followed by transfection of a mixture of the lentiviral shuttle plasmid, pMDG-VSVG, and pCMV-RD8.9 (a kind gift from Donald B. Kohn) using TransIT-293 (Mirus Bio) and OPTI-MEM (Life Technologies). Viruses were filtered through 0.45 micron syringe filters (Milipore) and stored at −80°C until further use. To transduce Jurkat, NFAT-GFP-Jurkat, or Primary T cells, 2-5×105 cells were plated in culture medium and mixed with an equal volume of thawed virus in 12-well plates for three days. For NFAT-GFP-Jurkat cells, G-418 was added to the transduction mixture 48 hours later.
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