Publication protocol
All the cell culture procedures below were performed at 300 K. Plasmid vector transfection to Drosophila S2 cells was performed by the calcium phosphate method according to the manufacturer's protocol (Invitrogen). Briefly, 9.5 μg of pAc‐mfT1r2aL(t), 9.5 μg of pAc‐mfT1r3L(t), and 1 μg of pCoBlast (Invitrogen) were co‐transfected to 3 mL of S2 cells (at the cell density of ∼2 × 106 cells/mL), cultured in complete Schneider's Drosophila medium (SDM, Invitrogen, supplemented with 10% FBS and 1 mM l‐glutamine) in a 35 mm dish or in a 6‐well plate. One day after the transfection, the cells were washed with and resuspended in fresh complete SDM. Three days after the transfection, the cells were resuspended in fresh complete SDM supplemented with 25 μg/mL Blasticidin S.
S2 cell cloning in soft agar was performed as basically described previously37 with some modifications. A week after the transfection, cells were plated in soft agar in order for antibiotic‐resistant cell selection and cloning. Typically, cells at three different amounts, 0.5 × 105, 1.7 × 105, and 5 × 105 cells, were resuspended in 9 mL of 75–50% fresh and 25–50% conditioned complete SDM supplemented with 25 μg/mL Blasticidin S, where the latter was prepared by filtration of the medium after the culture of nontransfected S2 cells for 2–4 days. Then, the cells were mixed with 1 mL of 3% low melting point agarose (Agarose‐LM, Nacalai Tesque) in PBS (10 mM NaH2PO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), which was completely melted by incubation at 353K, and plated in a 90 mm dish or two 60 mm dishes at the final cell density of 0.5 × 104, 1.7 × 104, and 5 × 104 cells/mL. The plates were put in a sealed plastic container for the prevention of dry up, and incubated for two weeks, avoiding vibration.
After the incubation (i.e., three weeks after the transfection), colonies of S2 cell clones were visible in size of ∼ 0.5–1 mm. A single colony was sucked up by a micropipette and plated in 100 μL fresh complete SDM with 25 μg/mL Blasticidin S in 96 well plates. Two to four days later, 100 μL of complete SDM was added to each well, and the cloned cells were subjected to further culture and expansion in the complete SDM.
The first high‐expression clone screening was performed generally a week after the S2 cell clones were plated in a 96 well plate. About 100 μL of the cell culture supernatant (the cultured medium) taken from each well was transferred to an Anti‐FLAG High Sensitivity, M2 Coated 96‐Well Plates (SIGMA). After 2 h of incubation at room temperature, each well was washed with 300 μL TBST (50 mM Tris, 150 mM NaCl, 0.5% Tween20, pH 7.4) three times, and the bound protein was eluted with 30 μL of the 2 × SDS‐PAGE sample buffer 125 mM Tris, 4% SDS, 20% glycerol, 0.004% bromophenol blue, pH 6.8, supplemented with 5 mM DTT). The second screening was performed after the clones selected at the first screening were expanded in a 24 well plate (with 0.5 mL culture volume) or in a larger scale. Typically, 200–500 μL of the cell culture supernatant taken from each well was mixed with 20 μL of ANTI‐FLAG M2 Affinity Gel (SIGMA) and rotated at 277 K for 1 h. After washing by TBS (TBS without Tween20) three times, the bound protein was eluted with 20 μL of the 2 × SDS‐PAGE sample buffer. In both cases, protein expression was analyzed by western blotting. The eluent protein was subjected to SDS‐PAGE with SuperSep Ace 10% gels (Wako) and electroblotted on a nitrocellulose membrane by iBlot (Invitrogen). After the blocking of the membrane by Blocking One (Nacalai Tesque), the protein was immunologically detected by use of Anti‐DDDDK‐tag HRP‐DirecT (MBL, Cat # PM020–7), and the chemiluminescent signals were detected using Immobilon Western HRP substrate (Millipore) and ChemidocXRS (Bio‐Rad).
The selected clones were adapted to a serum‐free medium, ExpressFive SFM, and subjected to protein production as described previously.31
For the establishment of a conventional stable cell pool (Suppporting Information Fig. S1), the cells transfected by pAc‐mfT1r2aL(t), pAc‐mfT1r3L(t), and pCoBlast as described above were passed in the presence of 25 μg/mL Blasticidin S for two weeks, according to the manufacturer's protocol.
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