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In Groups 2 and 3, the media containing cells and tissue was plated onto the same number of culture dishes as used in Group 1, either at high or low density.
A second layer (1ml) containing 0.3% of low melting agar dissolved in growth media containing cells (3×103 cells/ml) was placed on top of the first layer and allowed to set at 4°C for 20 minutes.
Differences in H2O2 levels were recorded in treated media containing cells and treated media only.
Before use, media was thoroughly vacuumed off and 200 microliters of media containing cells was added to the empty hole punch.
Tissue culture plates were coated with matrigel (BD Biosciences) diluted 1 200 in NEUROBASAL™ medium (GIBCO) for 1.5 hr at room temperature and washed with HyClone® DPBS/MODIFIED (1×) with calcium and magnesium (PBS++; Thermo Scientific) prior to application of media containing cells.
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For the AlmarBlue assay, 500 μL of culture media containing 10% of the AlamarBlue reagent was added into culture media containing cell-laden gels for 4 h.
To predict the effect of this IGF1 sequestration on cell proliferation, model equations were solved for two different scenarios, one corresponding to OVCAR5-conditioned media containing cell-secreted IGFBPs and one corresponding to fresh serum-free media in which no IGFBPs were present.
These sarcoma cells and all tumor cells were plated as a bead of media containing ~12,000 cells which results in a highly confluent area of cells as pictured in Fig. 1C.
Two hours after co-culture, media containing floating cells were discarded, and adherent cells were trypsinized.
Any media containing nonadherent cells were also collected and pooled with the trypsinised cells.
The media was removed from NP tissue explants and 50 μl Ad-GFP-infected MSC suspension (that is, 100,000 cells) was injected into tissue explants while 50 μl media containing no cells was injected into control tissue explants.
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