We've compiled some tips and instructions to consider implementing in your next ELISpot/FluoroSpot for working with adherent cells.
ELISpot and FluoroSpot are excellent methods to use when assessing the functionality of different adherent cells. There are some alterations that you should consider adding to your protocol though if working with these cells.
Tip!
Start with a healthy, confluent cell culture: Adherent cells should be grown in a healthy, confluent state before being used in an ELISpot assay. This ensures that the cells are in a proper physiological state and will respond appropriately to the assay.
Protocol:
- Detach cells from culture dish: Adherent cells must be detached from the culture dish by gently shaking or scraping the dish or flask. You can use trypsin-EDTA or other reagents like Accutase to detach the cells.
- After detaching the cells, it’s important to wash them thoroughly to remove any residual reagents or debris before adding them to the ELISpot/FluoroSpot plate.
- Count the cells and add the appropriate number of cells to the prepared ELISpot/FluoroSpot plate.
- Incubate the plate as instructed for your specific analyte of interest.
- Following incubation in the ELISpot/FluoroSpot plate, cells may be transferred for downstream analyses or discarded:
- Cell transfer: carefully remove the cells from the ELISpot/FluoroSpot plate and transfer to a new sterile 96 well plate. Wash the plate 3X with 200 µl PBS. Add 100 µl PBS with 1 mM EDTA and incubate for 10 minutes. Gently agitate the plate and resuspend the cells with a pipette before transferring and then washing the remaining cells.
- Cell discard: wash the plate 3X with 200 µl PBS and add 100 µl PBS with 1mM EDTA and incubate for 10 minutes. Wash the plate again 5X.
- Continue with ELISpot/FluoroSpot development steps.