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Astrocytes were analyzed in standard culture medium without starvation.
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This inhibition of SEACIT prevents the inactivation of TORC1 and entrance in autophagy upon switching of cells from rich to synthetic medium without nitrogen starvation.
We then transferred spheroids approximately 300 µm in diameter from free suspension into 1% agarose gel and cultured them under three conditions: normal medium without external compression, starvation medium (no glucose, no serum and 1% oxygen) without compression or normal medium with external compression (see experimental setup in Supplementary Fig. S1C).
Gene transcription profiles of cells that were treated only under nutrient starvation (10% Dubos medium-without glycerol at pH 7.0), was used as the internal control.
After attachment (4-h), cells were kept in starvation medium without dexamethasone and foetal calf serum.
Cells were then seeded in starvation medium (without antibiotics) containing 2nM 17β-ED or its vehicle.
NIH3T3 cell lines stably expressing different FlagHA-H3.3 species were seeded in selection medium containing 10% FBS and then starved for 2 days in starvation medium without FBS.
In starvation medium without serum, all transfectants migrated 60 100 μm during the 40 h period without any significant differences between experimental groups.
No growth and BS production was observed in the medium without any nitrogen source (nitrogen starvation) in the present study.
Briefly, tumor cells were grown in 15 cm culture dishes until 60~70% confluent, at which point the cells were exposed to the culture medium without FCS to induce serum starvation (to inhibit EGFR kinase activity).
The cells were then subject to overnight starvation with the medium without any FBS.
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