I am working with LM2 cells (a metastatic derivative of MDA-MB-231) that already have some baseline fluorescence, and I transfected them with GFP. after transfection i can see clear GFP expression under a fluorescence microscope, but during FACS sorting, I am getting a single peak, instead of a clear separation between GFP-positive and GFP-negative cells. Why.
Any suggestion
Thanks in advance
Himanshu ... , here's my recommdations:
Annie Huang
1. Baseline fluorescence interference: The autofluorescence of LM2 cells overlaps with the GFP signal.
- Use untransfected LM2 cells to calibrate the baseline, adjust the FITC channel voltage, and ensure that the negative control peak is in the low fluorescence area.
2. Small difference in GFP expression after transfection: The fluorescence intensity difference between positive cells and negative cells is insufficient.
- Extend the culture time after transfection (48-72 hours), or switch to higher brightness EGFP; optimize transfection conditions to improve efficiency.
3. Improper flow parameters: voltage and gain do not distinguish signals.
- Reduce the PMT voltage of the fluorescence channel to avoid "overflow" of negative cell signals; use scatter plots to exclude cell clumps (reduce false positives).
4. Sample processing problems: cell aggregation or debris interferes with peak shape.
- Remove clumps through a 40-70μm sieve, and resuspend in PBS containing EDTA after digestion to reduce adhesion.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
Hi — this is a common issue. A single peak can result from high baseline/autofluorescence, low or heterogeneous GFP expression, or suboptimal instrument settings. Make sure you run an untransfected (negative) control and a positive control, gate out debris/doublets and dead cells, use the FITC/GFP detector (488 nm, ~530/30), switch the histogram to log/logicle scale, and increase the PMT voltage so the negative sits left but not saturated. Overlay the negative and transfected histograms — if you still only see a small shift, your transfection may produce a continuum of dim expressors (microscope can make dim signal look obvious).
Solutions: improve transfection efficiency, use a brighter GFP variant (e.g., eGFP or mNeonGreen), or amplify signal with anti-GFP antibody. If you share your cytometer model, laser/filter set, and an example histogram, I can give more specific settings to try.
Dear Himanshu ...
I hope you have resolved your issue!
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