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To obtain probably higher recoveries, we transferred 100 μL aliquots of the supernatant of QC sample (10% TCA) onto a 96-well SPE method development plate (25 mg/well; Sigma Aldrich, Zwijndrecht, the Netherlands).
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After development, plates were dried in a stream of cold air for 10 min.
After development, plates were scanned and the peak area of each concentration and their Rf values were calculated.
To prevent progeny development plates were supplemented with 5-fluoro-2′-deoxyuridine (FUdR, 15 μM) on the day prior to use, unless indicated otherwise.
After development plates were dried in air and were placed for ca 5 min in a chamber containing chloride gas, excess chlorine was removed from the plates and the next plates were sprayed with o-tolidine reagent (50 mg o-tolidine in 100 mL 10% acetic acid).
Furthermore, similar to growth plate development in which hypertrophic maturation is under the regulation of a feedback loop involving Indian hedgehog and parathyroid hormone-related protein (PTHrP) [ 35], PTHrP also plays a regulatory role in MSC terminal differentiation.
Because the structural vertebral anomalies that caused the CS deformity may also affect growth plate development, it was inappropriate to consider the growth plates of the spine from CS patients as normal references.
This result suggested a dosage requirement for BMP/Smad signaling during growth plate development.
The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis.
Not surprisingly, BMP signaling components are highly expressed in growth plates with specific temporal-spatial patterns that correlate with functions during growth plate development and homeostasis.
In this regard, disruption of primary cilia results in abnormal skeletal patterning, post-natal growth plate development, and skeletogenesis [2], [3], [4], [5], [6].
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