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During classical cellular differentiation cells lose phenotypic plasticity until they become fully differentiated.
Twenty-four hours prior to differentiation, cells were passaged in LIF-containing IMDM medium.
To induce keratinocyte differentiation, cells were maintained in high-calcium medium (1.8 mM) for up to 72 hours.
To begin differentiation, cells (passages two through four) were switched to either osteogenic or adipogenic differentiation media and normoxic conditions.
To assess differentiation, cells cultured in differentiation medium were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde.
For smooth muscle differentiation, cells were cultured in the presence of 2% FBS and 10 ng/ml β-TGF for 4 6 days.
For neural differentiation, cells were cultured at high densities, spontaneously forming spherical clumps of cells that were isolated with 0.25% trypsin (Invitrogen).
To induce differentiation, cells were plated on dishes coated with 10 μg/ml laminin (Sigma-Aldrich) and cultured in NeuroCult Basal Medium containing NeuroCult Differentiation Supplement (Stemcell Technologies).
On day 3 of differentiation cells were treated with tBHP (200 μM) for 16 hours before an MTS assay was performed as described above.
To induce differentiation, cells were grown to 90% confluence and the media switched to DMEM supplemented with 2% horse serum (Hyclone, Fisher Scientific).
To determine if transient expression of Foxo13A can lead to persistent alterations in CD8+ T-cell differentiation, cells from three independent donors were treated with CD8-targeted Foxo13A-eGFP NPs, sorted based on eGFP expression, and maintained in vitro.
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