0.45 μm syringe filter

AGS-BX1 cells at 70% confluence were treated with the lytic-inducing compounds for 5 days or untreated. The culture supernatants were collected and centrifuged, then filtered with 0.45μm syringe filters (Sartorius Stedim Biotech, Goettingen, Germany). The filtered supernatants were used to superinfect Daudi cells as previously described [10 (link)]. The Daudi cells were analysed by flow cytometry (LSRII, BD Biosciences) for GFP expression to give an estimation of the level of infectious virus particles released into the culture supernatants upon compound treatment.

The sequences of variable heavy- and light-chain regions for the chosen antibodies were codon optimized and synthesized as eBlocks (Integrated DNA Technologies). The synthetic genes were cloned into a pcDNA3.4 vector (Invitrogen) containing human IgG1 heavy-chain and kappa or lambda light-chain constant regions, respectively (16 (link), 49 (link)). Heavy- and light-chain plasmids for each monoclonal antibody were cotransfected into Expi293F cells (Thermo Fisher Scientific). After incubating for 5 days at 37°C with 8% CO₂, the supernatant containing secreted antibodies was centrifuged at 4,000 × g for 15 min at 4°C, filtered through 0.45-μm syringe filters (Sartorius), and passed three times over a column with 1 mL protein G agarose resin (Pierce). After washing the column with 20 column volumes of PBS, antibodies were eluted with 10 mL 100 mM glycine-HCl (pH 2.7) and immediately neutralized with 1.5 mL of 1 M Tris-HCl (pH 8.0). Antibodies were buffer exchanged into DPBS, utilizing Amicon ultra-30 centrifugal spin columns (Millipore).

Lentiviral vectors for TCR expression were generated as previously described (56 (link)). Basically, the genes encoding TCR variable regions of TCR from single-cell V(D)J sequencing were fused with murine TCRα and β2 constant regions, respectively. The TCRα and β chains were linked by a P2A self-cleaving peptide. The TCR expression cassette was codon optimized, synthesized (GenScript, Nanjing, China), and cloned into the pRRLSIN.cPPT.PGK-GFP.WPRE lentiviral vector (Addgene, USA). TCR-encoding lentivirus were collected from 293T cells transfected with the transfer plasmid and the packaging plasmids PsPAX2, pMD2.G (Addgene, USA). 48 h and 72 h after transfection, cell supernatants were collected and filtered through 0.45 μm syringe filters (Sartorius, Germany). Virus particles were pelleted by ultracentrifugation (Beckman Coulter, USA), resuspended in T009, aliquoted on ice, and stored at -80°C.
To determine the viral infection titer, jurkat cells were infected with the lentivirus at different volumes, and the expression of exogenous TCR-β after transduction was detected by flow cytometry. The infection titer is calculated using the formula: Infection titer (IU/mL) = cell number at infection time × positive rate/virus volume.