Hi, I will be using a IFNg/IL2 Fluorospot Plus plate to detect antigen specific T cells. I will be using cells from Bronchoalveolar lavage fluid (BALF), which contains mostly macrophages that will adhere to the membrane during stimulation.
What would you suggest to use, to get rid of these adherent cells before addition of detection antibodies ? I am worried the adherent cells might lead to artifacts.
Thanks
Avinash
My experience from working with macrophages is that typically don't cause so much artefacts. The spots generated in ELISpot/FluoroSpot are rather large in comparison to the cells that gave rise to them. Atleast try and see what happens if you simply continue with the protocol as normal without removing adherent cells through some special protocol.
Nevertheless, you concern is noted. We have this recommendation:
- After you have incubated your cells overnight and it is time to remove them, wash as normal 5x with 200ul PBS.
- Incubate the plate in PBS containing 1mM EDTA in 37C for 10minutes.
- Take out the plate and wash again 5x with 200ul PBS.
After this you continue as normal with your detection protocol.
Hey Christian thank you for your prompt response.
Always happy to help out!