Flow Cytometry
Flow Cytometry Troubleshooting Tips
Source: Elabscience®Published: Jan 18,2024
1. Weak fluorescence signals or no fluorescence expression
Possible
causes
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Suggestions
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Improper storage or operation of antibody
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Store at 2~8℃ and protect from prolonged exposure to
light, avoid freeze/thaw circles.
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Fluorescence quenching
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Fluorescent antibody and sample added with
fluorescent antibody should avoid to light.
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High auto-fluorescence
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Change the fluorescent dyes to avoid dyes that emit
light the same with cell auto-fluorescence.
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Incorrect dyeing time or temperature
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Adjust the dyeing time and temperature properly.
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Use the wrong staining solution to stain
intracellular proteins
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For optimal intracellular protein staining, cell
fixation and membrane breaking should be performed using the correct fixation agent to detect
cytoplasmic and nuclear proteins.
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Secretory intracellular proteins
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For flow cytometry detect, secretory proteins such as
cytokines, chemokines, and growth factors must be retained in cells using blockers.
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Protein is down regulated, internalized, or shear cut
from cells
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Make sure that the stimulating conditions adopted
will not affect protein localization.
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The target protein is low expressed in the sample
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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.
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Antigens damaged by cell separation or frozen storage
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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.
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Abnormal performance of laser
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Use flow cytometer to set up and track (CS&T)
microspheres to check laser calibration and function.
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Incorrect use of filter
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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.
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Overcompensation of data
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Using single staining control and Fluorescence Minus
One (FMO) control to set compensation each time.
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Incorrect cell gate
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Ensuring the correct setting of cell group, use
reactive dyes and set up gate for single cell group can significantly reduce false positive.
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Incorrect data analysis
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In order to show the rare cells or dyed gloomy cells
excellently, dual parameters were used to observe the cells.
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2. High background of fluorescence/non-specific staining
Possible
causes
|
Suggestions
|
High auto-fluorescence
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Samples with the same stimulus condition but without
any staining are used as controls for auto-fluorescence.
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Antibodies bind to dead cells
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Use active dyes to exclude dead cells.
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Cy5 dyes
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Cy5 and other blue dyes will combine with some cell
Fc receptors, such as mononuclear cells and macrophages, please use other fluorescent dyes.
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High concentration of antibody
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Titrate the amount of antibody to make sure the
dosage of the antibody is suitable.
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The dyeing time is too long
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According to the expression of the cell protein,
optimizing the antibody concentration and incubation time.
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Insufficient washing
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Increase washing times after dyeing.
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Insufficient compensation regulation
|
Using single staining control and Fluorescence Minus
One (FMO) control to set compensation for the experiment.
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3. Fluorescence abnormality
Possible
causes
|
Suggestions
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Incorrect isotype control concentration
|
Use the same concentration of isotype control as
detection antibody.
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The isotype control and detect antibody from
different manufacturers
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Use the isotype control and the detection antibody
from the same manufacturer.
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Cell adhesion or dead cells are included in the
analysis
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Set up cell group gate and use active dyes to exclude
adhesion and dead cells.
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The immobilized and broken membrane fluid may affect
the cell characteristics
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Adjust the threshold to ensure that the cell
processes within the threshold range.
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Stimulation conditions change cell characteristics
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Cells are identified using marker anticyclic gate.
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