Common Problems and Solutions for Flow Experiments

1. No/weak staining

Possible causes	Resolvent
Improper storage and handling of antibodies	Antibodies should be stored at 2-8°C, protected from light, while avoiding repeated freezing and thawing.
Fluorescent dye quenching	Both fluorescent antibody and fluorescent antibody-stained samples should be stored away from light.
Higher autofluorescence	Changing the form of fluorescent dyes for antibodies, avoiding dyes that emit the same light as autofluorescence or compensate for it to a greater extent
Incorrect dyeing time and temperature	Adjust the dyeing time and temperature appropriately
Staining intracellular proteins with the wrong stain	For optimal intracellular protein staining, cells should be fixed and ruptured using the correct fixative rupture agent to detect cytoplasmic and nuclear proteins
Secretory intracellular protein	For flow cytometry, secreted proteins such as cytokines, chemokines and growth factors must be retained in the cell using blocking agents
Proteins are down-regulated, internalized or sheared from cells	Determine that the stimulus conditions used do not affect protein localization
Protein expression levels are too low	For antigens with low expression levels, the brightest fluorescent dyes are used for staining, and two-step staining can sometimes amplify the signal, e.g., using a biotinylated antibody followed by fluorescently labeled secondary antibody staining
Destruction of antigens by isolation protocols or frozen storage of cells	Enzymes used to collect cells from solid tissue or cell culture dishes can damage cell surface proteins; try preparing cells with non-enzymatic reagents. Test the reagents used to prepare the cells and the storage/handling of the cells for possible effects on antigenic
Instrument detection channel abnormality	Use flow cytometry QC microspheres to check the performance of each channel of the flow cytometer
Incorrect filter used	Check the excitation and emission wavelengths of the fluorescent dyes used to ensure that the correct lasers and filters are being used to collect the data.
Data overcompensation	Compensation was set for each experiment using a single-positive control and a fluorescence minus one (FMO) control
Incorrect gating of cell populations	Ensure proper gating of cell populations, use of dead-viable dyes and gating of single cell populations can significantly reduce false positives
Incorrect data analysis	For optimal display of rare cells or cells with low expression levels, a two-parameter plot was utilized to observe the cells

2. High background/non-specific staining

Possible causes	Resolvent
Higher autofluorescence	Samples using the same stimulation conditions but not stained with any reagents were used as controls for cellular autofluorescence
Antibody binding to dead cells	The staining includes staining with dead and alive dyes to exclude dead cells
Cyanine 5 dye	Cyanine 5 and other Cyanine 5 tandem dyes that bind non-specifically to Fc receptors on specific cells, e.g. monocytes and macrophages, consider using other fluorescent dyes
High antibody concentration	Dosage of titrated antibodies
Non-specific binding of secondary antibody/reagent	Titration of secondary antibodies to minimize background and block Fc receptors, ensuring the use of secondary antibodies that have been highly recognized as non-specific
Dyeing too long	Optimization of antibody concentration and incubation time based on experimental cell expression
Inadequate washing	Increase the number of washes after dyeing
Insufficient compensation regulation	Compensation was set for each experiment using a single-positive control and a fluorescence minus one (FMO) control

3. Abnormalities in coloring

Possible causes	Resolvent
Incorrect concentration of isotype control used	Use an isotype control of the same concentration as the test antibody
Isotype controls from different manufacturers	Use an isotype control from the same manufacturer as the antibody.
Inclusion of cell adhesions or dead cells in the analysis	Single-cell populations are gated and dead-viable dyes are used to exclude adhesions and dead cells
Fixation/breaking solution affects cellular properties	Adjust thresholds to ensure cells are within processing thresholds
Stimulus conditions alter cellular properties	Recognizing Cells Using Marker Reverse Circle Gates

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