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Flow Cytometric analysis

Flow Cytometry Troubleshooting Tips


1. Weak fluorescence signals or no fluorescence expression

Possible causes


Improper storage or operation of antibody

Store at 2~8 and protect from prolonged exposure to light, avoid freeze/thaw circles.

Fluorescence quenching

Fluorescent antibody and sample added with fluorescent antibody should avoid to light.

High auto-fluorescence

Change the fluorescent dyes to avoid dyes that emit light the same with cell auto-fluorescence.

Incorrect dyeing time or temperature

Adjust the dyeing time and temperature properly.

Use the wrong staining solution to stain intracellular proteins

For optimal intracellular protein staining, cell fixation and membrane breaking should be performed using the correct fixation agent to detect cytoplasmic and nuclear proteins.

Secretory intracellular proteins

For flow cytometry detect, secretory proteins such as cytokines, chemokines, and growth factors must be retained in cells using blockers.

Protein is down regulated, internalized, or shear cut from cells

Make sure that the stimulating conditions adopted will not affect protein localization.

The target protein is low expressed in the sample

For the antigen with low expression, the brightest fluorescent dye must be used in dyeing. Sometimes two step staining can improve sensitivity, for example, using biotin-Labeled antibody first, then fluorescent labeled secondary antibody staining was used.

Antigens damaged by cell separation or frozen storage

Enzyme used to collect cells from solid tissues or from cell dishes may destroy surface proteins, so try to prepare cells with none enzymatic reagents,and check the reagents used to prepare the cells and the storage/handling of the cells to make sure the reagents will not affect antigens.

Abnormal performance of laser

Use flow cytometer to set up and track (CS&T) microspheres to check laser calibration and function.

Incorrect use of filter

Check the excitation wavelengths and emission wavelengths of the fluorescent dyes to make sure that the correct laser and filter used to collect the data.

Overcompensation of data

Using single staining control and Fluorescence Minus One (FMO) control to set compensation each time.

Incorrect cell gate

Ensuring the correct setting of cell group, use reactive dyes and set up gate for single cell group can significantly reduce false positive.

Incorrect data analysis

In order to show the rare cells or dyed gloomy cells excellently, dual parameters were used to observe the cells.

2. High background of fluorescence/non-specific staining

Possible causes


High auto-fluorescence

Samples with the same stimulus condition but without any staining are used as controls for auto-fluorescence.

Antibodies bind to dead cells

Use active dyes to exclude dead cells.

Cy5 dyes

Cy5 and other blue dyes will combine with some cell Fc receptors, such as mononuclear cells and macrophages, please use other fluorescent dyes.

High concentration of antibody

Titrate the amount of antibody to make sure the dosage of the antibody is suitable.

The dyeing time is too long

According to the expression of the cell protein, optimizing the antibody concentration and incubation time.

Insufficient washing

Increase washing times after dyeing.

Insufficient compensation regulation

Using single staining control and Fluorescence Minus One (FMO) control to set compensation for the experiment.

3. Fluorescence abnormality

Possible causes


Incorrect isotype control concentration

Use the same concentration of isotype control as detection antibody.

The isotype control and detect antibody from different manufacturers

Use the isotype control and the detection antibody from the same manufacturer.

Cell adhesion or dead cells are included in the analysis

Set up cell group gate and use active dyes to exclude adhesion and dead cells.

The immobilized and broken membrane fluid may affect the cell characteristics

Adjust the threshold to ensure that the cell processes within the threshold range.

Stimulation conditions change cell characteristics

Cells are identified using marker anticyclic gate.

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