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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
Hi Mabtech! I used your human IgG/IgA/IgM FluoroSpot kit (FS-050617) to look at total and vaccine specific plasmablasts after vaccination with different licensed vaccines. Overall the kit and protocol was well constructed and easy to follow! I used cryopreserved cells that I rested for 1 hour before moving them to the plates for incubation. The plates were coated with either the anti-IgG/A/M included in the kit or 10 µg/ml of inactivated whole virus (specific to the vaccine). The antigen specific wells didn't seem to work at all, but the highest concentration of cells worked well with the total IgG/A/M and are similar to our FACs data from these time points. However in certain wells there is really high background. I used a 12-channel multichannel pipette so I would expect that all the wells in the row to look somewhat similar, but they don't seem to be that way. Any ideas why this could be? Thanks! Fluorospot IgG:IgA:IgM.pdf