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Both LRP and LR were scored separately but for the initial set of experiments (Figs 1 and 2) the LRP and LR are combined for simplicity.
The CMV promoter was used in the initial set of experiments as it sensitive to position effects in ES cells and although the results described apply only to this promoter, work is currently underway to test a range of neuron specific promoters to determine how efficiently these work when targeted to the ROSA26 locus.
As the current study was more focused on basic pathophysiological mechanisms and not (yet) therapeutic interventions, we selected a model without antibiotics for the initial set of experiments.
The study endpoint of the initial set of experiments was the relationship between apoptosis of lymphocytes at 4 and 24 hours with 7-day survival.
For the initial set of experiments, relatively high concentrations were applied (10 40 μM for DOX and 16 128 μM for cisplatin) and crystal violet staining was used to monitor cell viability following treatment for 20 h and 1(b)).
For the initial set of experiments screening for the loss of a PstI restriction site situated between the PB1 recognition sites, the PCR amplified fragments were digested overnight at 37°C with 20 U PstI (New England BioLabs) in 1x NEbufferfer.
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Since the location of this area was very consistent across animals in a set of initial experiments (n = 58 rats, with not all initial experiments yielding data points), intrinsic imaging was performed only in this initial set of experiments and in the following experiments, neurons were targeted stereotactically in this location.
Finally, in this initial set of experiments, MV4-11 cells were treated with the CNDAC/vorinostat combination using the three treatment schedules evaluated in the cytotoxicity experiments to ascertain whether the sequence of compound administration had a significant influence on the amount of cell death.
Anne-Marie Lüchtenborg performed most of the experiments and wrote the paper, Vladimir Purvanov performed site-directed mutagenesis and performed the initial set of some experiments, Bogdan Melnik contributed to the design of the FRET experiments, Simon Becker performed molecular modelling, and Vladimir Katanaev conceived and designed the experiments, interpreted the data and wrote the paper.
In the initial set of small-scale experiments, a vertical 1 cm diameter solid aluminum cylinder with varying lengths is placed centrally in a 10 cm diameter pan fed with a continuous supply of hexane burning in quasi-steady state.
In the initial set of whole-cell experiments, 0.5 1 mM choline, a selective endogenous agonist of α7 nAChRs, was focally administered to the recorded hippocampal CA1 pyramidal neurons via a picospritzer pipette identical to that used for patch-clamp recordings.
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