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.
Costar 96 well cell culture plates
Manufactured by Corning
The Costar 96-well cell culture plates are designed for in vitro cell culture applications. They provide a standardized format with a 96-well configuration to facilitate efficient cell growth and analysis.
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4 protocols using costar 96 well cell culture plates
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.
4
Assessing Relative Fitness of Yeast Strains
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To measure relative fitness we used an automated high-resolution method based on fluorometry previously developed by (DeLuna et al. 2008) . The mCherry-tagged collection carrying individual 'duplications' (Y8205-mCherry +MoBY-xxx) were competed with a universal CFP-tagged strain (Y7092-CFP +p5586). First, we inoculated 150 µL of fresh medium (using Corning® Costar® 96well cell culture plates) with overnight cultures of 'duplicated' and reference strains (tagged with mCherry and CFP respectively) in a 1:1 proportion. Every 24 h, we diluted the cultures 16-fold into sterile fresh competition media. Cultures were monitored in parallel for approximately 28 generations (7 days) in a fully automated robotic system (Freedom EVO, Tecan Ltd) connected with a microplate multi-reader (Infinite Reader M100, Tecan Ltd). We monitored OD600 nm, mCherry fluorescence signal (578/610 nm, gain: 145), and CFP fluorescence signal (433/475 nm, gain: 120) every 2 h. Cultures were incubated at 30 °C and 70 % relative humidity.
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