Biological control (e.g., unstimulated sample in stimulation assay) shows unexpectedly high background, making it difficult to set clear positive/negative boundaries.
Common Causes
1Unstimulated sample shows elevated baseline expression of the marker being studied
2Cells in biological control sample were inadvertently activated during handling or preparation
3High autofluorescence or endogenous marker expression in the control cell population
4Contamination or carryover from experimental samples into control sample
Solutions
1Carefully handle biological control samples to avoid inadvertent cell activation during preparation
2Verify that control samples are truly representative of negative expression state
3Consider using FMO or isotype controls in addition to biological controls when high background is observed
4Optimize cell preparation protocols to minimize autofluorescence and maintain cells in unstimulated state
5Use freshly prepared biological controls and process them identically to experimental samples except for the variable being tested