96 well plate was seeded with 10000 cells/well. Compound pre-treatment was given to the cells at 10 uM, after which LPS treatment was given. Alamar assay showed similar fluorescence reading for both CONTROL and LPS experimental group. Now I am not able to infer which all compounds are safe (since my reference i.e. LPS is showing similar reading as CONTROL). Any help would be much appreciated.
Hi, treat your cells along with LPS. If the test substance reduces the fluorescence signal compared to the LPS-only control, it suggests that the substance is cytotoxic at that concentration. If the fluorescence signal is similar to the control, it suggests the substance is not cytotoxic.
Hello Varun,
Pretreatment with a compound can modulate cellular pathways involved in the LPS response, effectively reducing the subsequent inflammatory response triggered by LPS. Pre-treating RAW 264.7 macrophage cell line with the compound will allow them to engage with their targets before LPS can fully activate the inflammatory response, thus mitigating its effects.
In your experiment, it is crucial that you include two controls.
1. LPS-only control: This control group will allow you to see the typical inflammatory response induced by LPS which will provide a reference point to compare the effects of the pretreatment.
2. Pretreated cells without LPS control: This control group will help you to determine if the pretreatment itself has any unintended effects on the cells. For instance, it could be cytotoxic or alter cell behavior in a way that might confound the results.
Best,
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