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To investigate the binding of AP-tagged proteins to Lrp4 in a cell-free system, media containing of the Lrp4 ectodomain fused to the constant region of Immunoglobulin G (Lrp4ecto-Fc) was produced by transfection of HEK293A cells with pcDNA3-LRP4ecto-Fc in DMEM plus 0.2% BSA medium.
In contrast, CC109 cells transformed with a plasmid encoding GstB could grow in the presence of media containing of up to 3 mM sodium arsenate.
Cells were resuspened in 1 ml media containing of JC-1 dye (2 μM), and incubated at 37 °C, 5% CO2 for 40 min.
Each well was filled with 500 μl of media containing of MTT-reagent, consisting of 5 mg/ml MTT (Carl Roth) in PBS.
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A volume of media containing 50ng of the sECD was added to a final volume of 1 mL of serum-free media and placed in the bottom chamber of a modified Boyden chamber assay.
In a different set of experiments, CD36+ cells were cultured in liquid media containing one of each of the three ligands.
Anaerobic encapsulated cultures were started by transferring 30 capsules to 120 ml of media containing combinations of glucose (0 or 40 g/l), xylose (0 or 40 g/l) and furfural (0, 1 or 2 g/l) (Sigma-Aldrich).
The medium was removed after 24 h of incubation and a solution of fresh media containing 20% of CellTiter 96® AQueous One Solution reagent was added.
For the AlmarBlue assay, 500 μL of culture media containing 10% of the AlamarBlue reagent was added into culture media containing cell-laden gels for 4 h.
After 72 hours 100μl of growth media containing 10μl of CCK8 (Promega, Madison, USA) was added to each well for 3 hours at 37°C.
Therefore the medium that contained SmAQP-suppressed worms simply looked redder than media containing either of the control groups of worms.
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