There are many light, fuzzy, small dots in the background, are they the target cells I need? If not, why would these dots appear in the background? and what kind of dots represent the target cells?
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The Light, fuzzy, small dots in ELISPOT
- GRGuest Rain2025-03-17T02:59:47Z
- ESEsther Schoutrop2025-03-21T10:31:43Z
Hej there Rain,
The spots generated in ELISpot assays are the footprint of cytokine release by immune cells during incubation. It is normal to have spots of different sizes and intensity, as this is dependent on cytokine production by each cell.
Small and faint spots can be caused due to suboptimal experimental settings and cell handling steps, therefore it is important to include a negative control consisting of cells alone + medium to account for any background reactivity. Is one of the attached images the negative control consisting of cells alone + medium? Without having all the experimental information, it is difficult to give the most suitable answer and therefore I am addressing some pointers below that might not all be applicable to you:
- Suboptimal coating of the ELISpot plate with the capture mAb can result in fainter and fuzzy spots. Were these ELISpot flex plates or precoated plates? Coating of ELISpot plate tutorial: https://www.youtube.com/watch?v=c0lC3CiIB5A.
- Background reactivity due to ‘spontaneous release’ of cytokines which can be the result of the serum batch, suboptimal cell handling resulting in poor viability and high frequency of apoptotic cells or in vivo activation amongst others.
- Resting of cells following isolation has been shown to decrease background reactivity and enhance the signal to noise ratio: https://www.mabtech.com/knowledge-hub/why-resting-cells-essential-accurate-elispot-and-fluorospot.