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The filtered growth medium sample was thawed and 360 μl of the filtrate and 40 μl of either S. marcescens or N. capsulatum inoculum (OD = 0.25) was inserted into each well of the microtiter plate (Honeycomb 2, Thermo Electron Oy).
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Albumin concentration in the collected medium samples was analyzed using a competitive enzyme-linked immunosorbent assay [ELISA].
Albumin concentration of medium samples was analyzed by an enzyme-linked immunosorbent assay (ELISA), in triplicate, utilizing a polyclonal antibody to rat albumin (Cappel Laboratories, Aurora, OH).
Urea concentration of medium samples was determined via its specific colorimetric reaction with diacetyl monoxime using a commercially available assay kit (BUN Assay kit; Stanbio Laboratory, Boerne, TX) [29], [30].
After inoculating the isolated tetrads or microspores in modified NLNS medium, sampling was performed at 1-, 3-, 5- and 7-day intervals.
Medium samples were collected during a complete medium exchange every 48 h for glucose, insulin and albumin analyses.
Cells were incubated for 24 h before medium samples were collected for cytokine measurements and for 4 h or 24 h in case of intracellular protein analyses.
At the end of the experiment (on day 31), porous medium samples were destructively collected and analyzed for abundance of total and active bacterial cells, bacterial cell volume and concentration of polysaccharides and proteins.
Cell culture medium samples were collected every 2 h from 8 h post-induction.
Medium samples were taken by filtration of 2 ml of the culture broth through a 5-micron pore size nylon filter.
The culture medium was replaced every 24h and medium samples were stored at −20°C for further analysis.
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