Empty/negative control also shows fluorescence (high false-positive background) (Transfection)

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Untransfected or empty-vector wells show a clearly green/red signal — you can't tell if the target plasmid is really expressing. Common causes: 1) Cells autofluoresce / culture medium has high background 2) Microscope exposure or gain too high 3) Reagent color or fluorescent dye contamination 4) Vector contamination or insufficient washing Solutions: 1) Always include an untransfected blank AND an empty-vector control 2) Lower exposure and gain; standardize imaging parameters across groups 3) Verify imaging channel settings to avoid bleed-through 4) Change to fresh medium and wash more thoroughly before imaging