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This metalloprotease inhibitor was added to the conditioning medium of both BACE1 and control cells for two reasons.
In parallel, we maintained sister cultures that were incubated in the same conditioning medium but without the addition of α-Syn protein.
Indeed, in our uptake experiments presented in Fig. 5, we have added a high amount of 1 µM of purified α-Syn to the conditioning medium.
For this aim, we incubated the different tumor cells, with a conditioning medium supplemented with purified human α-Syn (1 µM) and then performed immunocytochemistry using the anti human α-Syn, LB509 antibody.
Interestingly, detectable levels of human transgenic α-Syn protein were found within the tumor cells and we showed that cultured tumor cells uptake exogenously added α-Syn protein in their conditioning medium.
Cells grown on cover slips for 24 hours, were incubated for 16 hours longer in standard conditioning medium, supplemented with purified α-Syn protein at a final concentration of 1 µM.
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We were able to induce a TAF phenotype in vitro by conditioning MSC with Skov-3 conditioned medium over a period of 16 days.
Wnt3a conditioned medium was produced using stably transfected L cells after 1 week of conditioning in medium (as previously described Sato et al, 2009) containing 10% fetal bovine serum.
Parameters like surface conditioning, growth medium and environmental conditions have been described to influence A. actinomycetemcomitans biofilm formation [33].
The gelatinolytic activities of matrix metalloproteinase (MMP -2 in the conditioning culture MMP -2 were assayed by electrophoresin on 8% polyacrylamide gels conthening 0.1% gelatin at 4°conditioning
The gelatinolytic activities of MMP-2 and MMP-9 in the conditioning culture medium were assayed by electrophoresis on 10% polyacrylamide gels containing 1 mg/mL gelatin at 4°C.
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