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Further, we analyzed different lengths of incubation of virus mixed with serum.
The wash buffer was decanted and the standards, assay buffer, or serum samples were mixed with serum matrix in each well.
For all groups, serum was isolated by centrifugation at 3000 rpm for 10 min after standing at room temperature (RT) for 30 min, and then mixed with serum from the same group.
The beads were mixed with serum samples (diluted 2-fold), incubated with shaking for 2 hours at room temperature, washed, and incubated with a cocktail of 14 different biotinylated detection antibodies for 1 hour.
In 1875 German physiologist Leonard Landois showed that, if the red blood cells of an animal belonging to one species are mixed with serum taken from an animal of another species, the red cells usually clump and sometimes burst i.e., hemolyze.
For PT assay, samples (25 μL) were mixed with serum (25 μL) and incubated at 37 °C for 3 min.
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Thrombin was reconstituted in sterile water containing 0.1%% (w/v) bovine serum albumin, mixed with serum-free culture medium and added directly to the wells in a concentration of 5 U/ml [ 14, 54].
In the invasion assay, the upper chamber of the transwell inserts with 6.5-mm polycarbonate membranes with 8.0-μm pores (Corning) was coated with Matrigel mixed with serum-free medium (diluted at 1 5) (BD Biosciences).
LPS was reconstituted in sterile water and mixed with serum-free medium and added directly to the wells in a concentration of 0.1 μg/ml as previously described [ 70, 71].
4T1 cells were seeded in a 96-well plate (800 cells per well) with GC of various concentrations (0, 50, 100, 150, 200, and 250 μg/ μl, and incubated at 37 °C for 4 days. After removal of the supernatant, 1 mg/ml MTT solution (Sigma-Aldrich Co). was mixed with serum-free medium and added to each well.
As a control, serum #32 was mixed with normal serum and the resultant bispecific signal was extremely low (mean OD: 0.045, data not shown).
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