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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.
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.
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.
The extraction media containing the cell suspension was centrifuged at 300 g for 20 min. Cell pellets were re-suspended in 30 ml of basal medium before mechanical trituration through 10 ml and 5 ml pipettes until cells were separated.
Virus containing cell media was collected after 48 hours of infection.
For IP experiments, cells were incubated with either vehicle (0.5% DMSO) or with indicated concentrations of GW2974 in 0.5% DMSO containing cell media for 2 h.
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.
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