1) Yes, we stimulated the PBMC for 3 days with R848 (0.5ug/ml) + IL2 (5ng/ml), washed and added to plate. 2) 2x10^5 cells for PBS and antigen coated wells. 1x10^4 cells for Total IgA/G/M I understand that IgM is 'sticky' due to its pentameric structure and binding, but we really want this to work as it will complement out optimised IgA, IgG and IgM ELISAs Are there any further tips you could suggest ?
Hi Christian, We are currently experiencing high background with the IgM detection within our IgA/G/M fluorospot. This issue seems to present wherever cells have been plated IgA and IgG detection works well ! We have followed the Mabtech IgA/G/M fluorospot kit protocol exactly Any ideas on how the background (non specific binding) for IgM-cy5 can be reduced ? Attached are our plate fluorospot read outs and plate plan Thanks Fluorospot issue Cy5.pptx