What to do if single stain control using endogenous reporter do not show good positive peak
Narmadha Murugesan A few quick thoughts:
All the best!
Sidney T. Sithole Okay. I will try these. Thanks for the response.
Narmadha Murugesan , Sidney T. Sithole
Strategies for Achieving a Good Positive Peak in Single Stain Controls for Rare Cell Markers.
1. Maximize the Population for Controls
May you nedd process as much starting material as possible to enrich for rare cells and ensure enough positive events are detected for your single stain control. Otherwise, use pre-enrichment techniques (e.g., magnetic bead sorting or density gradients) to boost the frequency of your cell type of interest in the control sample.
2. Optimize Antibody Staining
It's important to titrate each antibody to use at its optimal concentration—excess can increase background, while too little gives a weak signal.
It's recommend that use fresh aliquots, especially for conjugated antibodies, to avoid loss of signal due to degradation, particularly with tandem dyes.
3. Control Nonspecific Background
And you can use Fc receptor blocking reagents to minimize nonspecific antibody binding. Don't forget to stain with viability dyes to exclude dead cells, which can bind antibodies nonspecifically and increase background[9].
4. Others
When rare cell events make collecting enough positive cells impossible, use compensation beads. These beads coated with anti-IgG can robustly bind the antibody and provide a clear, bright peak for single-stain controls.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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