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The medium samples collected at days 3 and 6 were stored at -20°C.
Melatonin concentration in the medium samples collected during the experiments I IV was measured by a direct RIA with the use of G/S/704-6483 andibody and H-melatonin, according to the previously described and validated procedure [ 32].
To examine if the Leu69Ala and Leu69Lys substitutions in fusion proteins, namely TH-ProCLV3Leu69Ala and TH-ProCLV3Leu69Lys, respectively, led to compromised cleavages, Q-Tof MS/MS analyses were performed on medium samples collected after co-cultivations of these fusion proteins with wildtype seedlings.
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For recording conidial and mycelial differential interference contrast (DIC) images, cultures were performed in liquid minimal medium and samples collected at indicated time points.
Immediately after irradiation, cells were resuspended in liquid medium and samples collected at various time points – taken between 0 h and 5 h after irradiation – were analyzed.
The remaining culture was then collected by filtration, transferred to the appropriate medium after one wash with 100ml of -Glu medium, and further samples collected in TCA, as described above.
Medium samples were collected during a complete medium exchange every 48 h for glucose, insulin and albumin analyses.
Cells were incubated for 24 h before medium samples were collected for cytokine measurements and for 4 h or 24 h in case of intracellular protein analyses.
Cell culture medium samples were collected every 2 h from 8 h post-induction.
The culture medium was replaced every 24hr and medium samples were collected and stored −20°C until further analysis.
Culture medium samples were collected after 24 hr, centrifuged and the supernatant in each group was used to measure the levels of MCP-1 and MIF with an ELISA kit, following the manufacturer's instructions (R&D Systems, MN).
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