Hello Fatma Mohamed Fouad

The control cells are untreated cells, and therefore, they are assumed to have 100% viability. So, they should show a high level of alamar blue reduction and, consequently, a high optical density (OD) reading.

How high should be the OD reading (actual value) in the control is something very difficult to answer because the OD value will vary depending on factors like cell type, incubation time, and the specific alamar blue reagent that you use. However, the control cells should exhibit a significantly higher OD than background readings (medium without cells) and ideally, a higher OD than treated cells.

Best,

An OD of 0.5 in control cells using the Alamar Blue assay after 3 hours can be acceptable depending on the cell type, metabolic activity, seeding density, and plate reader settings, but ideally, the signal should be in the linear range of detection to ensure sensitivity to changes induced by treatment. If increasing cell numbers does not improve the OD significantly, consider the following:

• Extend the incubation time to 4–6 hours to allow more reduction of resazurin to resorufin.
• Ensure proper plate reader settings (usually fluorescence at Ex/Em 560/590 nm is more sensitive than absorbance at 570/600 nm).
• Confirm that cells are healthy and metabolically active, as stressed or dying cells may underperform.
• Use a standard curve of cell number vs OD to validate assay linearity.

If your OD consistently stays around 0.5 despite optimizing these parameters, you may need to increase either the incubation time or switch to fluorescence reading for better sensitivity.