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However, there is presently no fast, reliable method for measuring these rates for pilot and production scale high pressure homogenizers.
Measuring these rates is easy when the sample is known and knowledge of these rates is important to ensure that the validation tool does not introduce a bias.
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We measured these rates of evolution in two different ways.
Na22 flux could be used to measure these rates if desired, or a more quantitative pH probe, like pyranene.
When constructing the models we used in vitro rates as it is very difficult, if not impossible to measure these rates in vivo.
It was also not possible to measure these rates directly because of the absence of polymorphisms between the chromosomes we used due to both being inserted into the same parental landing site chromosome.
In addition, once it becomes possible to measure these rates within single cells, the predictions of the model presented here may be tested experimentally and used to improve the model.
Measuring these low rates was a daunting and laborious task that in the end required hundreds of liters of media and the screening of hundreds of thousands of colonies.
This method is based on dynamic flux of hydrogen and oxygen through the body and ability to measure these flux rates over a period of time using labeled isotopes, H and O [ 17].
This is accomplished by measuring the rate at which these cellular antioxidant molecules inhibit the metmyoglobin-catalyzed oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) to its radical cation form.
By independently measuring these three processes under identical experimental conditions, we were able to readily decouple internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion by simply measuring the lag between the measured rate curves for these three processes.
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