Exponential growth-phase adherent HEK293 cells were re-suspended in DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 1 mM sodium pyruvate, 0.1 mg/mL streptomycin and 10 % ultra-low IgG foetal bovine serum (FBS) (Thermo Fischer Scientific) and seeded at 4 x 104 cells/well in 100 μL 24 h prior to transfection in Costar 96-well cell culture plates (Corning). On the day of transfection, for each well, 50 μL of 60 μg/mL linear 25 kDa PEI (Alfa Aesar) was mixed with 200 ng of cognate heavy- and light-chain coding plasmid in a volume of 50 μL and shaken at 20°C for 30 min. The DNA-PEI complexes were added to the HEK293 cells. The following day, an additional 50 μL of supplemented DMEM (as described above) was added to each well. Supernatants were screened for PvDBPII (produced in S2 cells as described below) binding by indirect ELISA. Over 100 mAb supernatants were screened in this assay and of these, ten bound PvDBPII.
Linear 25 kda pei
Manufactured by Thermo Fisher Scientific
Linear 25 kDa PEI is a transfection reagent for DNA and RNA delivery in cell culture applications. It is a polyethylenimine (PEI) polymer with a molecular weight of 25 kilodaltons. PEI is a cationic polymer that can form complexes with nucleic acids and facilitate their uptake into cells.
Automatically generated - may contain errors
3 protocols using linear 25 kda pei
1
Antibody Screening and Production in HEK293 Cells
2
Antibody Screening and Production in HEK293 Cells
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Exponential growth-phase adherent HEK293 cells were re-suspended in DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 1 mM sodium pyruvate, 0.1 mg/mL streptomycin and 10 % ultra-low IgG foetal bovine serum (FBS) (Thermo Fischer Scientific) and seeded at 4 x 104 cells/well in 100 μL 24 h prior to transfection in Costar 96-well cell culture plates (Corning). On the day of transfection, for each well, 50 μL of 60 μg/mL linear 25 kDa PEI (Alfa Aesar) was mixed with 200 ng of cognate heavy- and light-chain coding plasmid in a volume of 50 μL and shaken at 20°C for 30 min. The DNA-PEI complexes were added to the HEK293 cells. The following day, an additional 50 μL of supplemented DMEM (as described above) was added to each well. Supernatants were screened for PvDBPII (produced in S2 cells as described below) binding by indirect ELISA. Over 100 mAb supernatants were screened in this assay and of these, ten bound PvDBPII.
3
Transient Expression of PfRH5FL in HEK293T
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Exponential growth-phase adherent HEK293T cells were resuspended in DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% ultra-low IgG FBS (all from Thermo Fisher Scientific) and seeded at 4 × 104 cells/well in 100 μL 24 h prior to transfection in Costar 96-well cell culture plates (Corning). On the day of transfection, for each well, 50 μL of 60 μg/mL linear 25 kDa PEI (Alfa Aesar) was mixed with 200 ng of cognate heavy- and light-chain coding plasmid in a volume of 50 μL and shaken at 20°C for 30 min. The DNA-PEI complexes were then added to the HEK293T cells. The next day, an additional 50 μL of supplemented DMEM (as described above) was added to each well. Supernatants were screened for PfRH5FL binding by indirect ELISA as described in the ELISA methods section.
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