1. No fluorescent signal
Antibody causes
1. Ensure that the antibody is preserved in accordance with the antibody instructions, pay attention to the preservation conditions, such as the use of PE/APC and other fluorescent labeled antibodies, should avoid freezing and thawing of antibodies, PE-cy5 dyes should avoid light, etc.

2. Antibody shelf life and antibody use concentration is too low, resulting in low or no signal
3. For some low-expression target proteins, antibodies with strong fluorescent signals should be used to avoid the use of secondary antibodies leading to no signal.
4. Check the use of appropriate fluorescent-labeled antibodies, distinguished from WB, IP selected FACS-specific antibodies

5. The primary antibody and the secondary antibody cannot recognize each other. If the primary antibody selects murine antibodies and the secondary antibody selects antibodies of other species to recognize, the secondary antibody and the primary antibody cannot combine with each other without signal
Sample and Reagent Problems
6. Conformational change of sample protein
The sample is fixed, protease digestion and other changes the natural conformation of the cell protein, resulting in the fluorescent antibody can not be combined, should pay attention to the use of fluorescent antibody precautions

7. The research target exists in the cell, and the membrane is not broken or broken enough, resulting in the antibody can not be combined. Small molecular weight fluorescent dyes should be selected to increase the binding of antibodies and targets

8. The antigen expression of the protein itself is low, or it is not expressed, or it needs to be expressed after induction by specific factors, etc. The protein is a secreted protein, which does not exist in the cell membrane, the extracellular region of the protein is short, and the fluorescent antibody cannot recognize the specified site; The target protein is internalized, and attention should be paid to incubation and storage at 4 ℃. The literature can be consulted or preliminary experiments can be done to confirm the abundance of target expression of the sample

9. Select the appropriate buffer, for example, some transcription factors may be sensitive to PH, ions, etc., should choose the appropriate buffer
Experimental process issues
10. Fixation time is too long, resulting in protein cross-linking and affecting the chemical properties of fluorescein

11. Cell samples are compensated with appropriate gain to ensure suitable detection conditions such as voltage, channel, etc.

12. Adjust the cell density and antibody incubation time to avoid using too many cells or insufficient incubation time.

13. Fluorescent reagents are destroyed by immobilized reagents, for example, methanol and PE dyes should be avoided

14. Excitation wavelength use error
Ensure that the excitation wavelength matches the fluorophore, the channel and fluorescence compensation match, etc.

2. non-specific staining
1. Sample autofluorescence
Different cells, media, and buffers may cause different autofluorescence. An unstained control group should be set to exclude fluorescent signals from the sample.

2. cell activity
Some dead cells have relatively high spontaneous fluorescence, which may lead to abnormal analytical signals. Dead /live cell dyes can be selected to distinguish

3. Antibody use conditions are not appropriate
The use of antibody concentration is too high will cause the background value to deepen and non-specific staining. It is recommended to operate in strict accordance with the recommended concentration of the instructions, and the concentration titration before the experiment can effectively control the signal-to-noise ratio. 

4. Sample with FcR
The binding of the Fc end of the antibody to cells leads to non-specific staining. When analyzing cells of myeloid origin, Fc blocking reagents can be used for blocking, but it should be noted that there is no cross-reaction between the blocking reagents and the secondary antibody. While avoiding the use of anthocyanins as fluorescent dyes to analyze FcR-expressing cell samples

5. Insufficient washing
Consider increasing the number of immersions after antibody incubation and extending the immersing time to avoid non-specific binding caused by insufficient cleaning times or time.

6. Non-specific staining may be caused by non-specific binding of secondary antibodies. It is recommended to add additional samples and secondary antibodies as experimental controls, and select a secondary antibody that only binding with primary antibodies but does not binding with detected cells.

7. Compensation setting inappropriate
Fluorescence overlap occurs between multiple fluorophores, and appropriate voltage and compensation should be adjusted. For some cells with autofluorescence labels, attention should be paid to adjusting the compensation between different fluorescence signals

3. High background

1. Some dead cells have relatively high spontaneous fluorescence, which may lead to abnormal analytical signals. Dead /live cell dyes can be selected to distinguish

2. When analyzing some special cells, such as granulocytes, the sample autofluorescence is relatively high.

3. Use recommended antibody dilutions. When using a lower cell number, the concentration can be determined by titration before the experiment.

4. It is recommended to use secondary antibodies with good specificity and clear recognition sites to avoid non-specific binding of fluorescent dyes or fluorescent antibodies directly to cells.
5. Incorrect optical parameter settings may result in insufficient signal strength or high background noise

4. Direction-finding light anomaly
1. Cell disruption
During the experiment, attention should be paid to vortex and centrifugal speed, permeabilization fixation time, etc., to avoid serious sample cell fragmentation.

2. Sample contamination
Improper preservation of samples, resulting in contamination of particulate matter containing bacteria, resulting in rapid sample collection, background autofluorescence, etc.

3. When dealing with blood products, the sample may contain red blood cell fragments, platelets and other impurities, and the washing steps can be appropriately optimized to reduce the content of impurities.
5. Weak Fluorescence Signal
1. insufficient antibody binding
If the antibody does not bind enough, the signal on the cell will be reduced. Possible causes include too low antibody concentration, insufficient binding time, etc.

2. Fluorescent labeled antibody signal is weak
Improper storage of fluorescent secondary antibody leads to weakening of fluorescence intensity, and the fluorescent marker substance may be unstable or has expired, resulting in weakening of signal intensity

3. Improper setting of photomultiplier tube 
The photomultiplier tube in the flow instrument is responsible for detecting the fluorescence signal, and its sensitivity setting affects the signal intensity. 

4. Insufficient cell usage
The signal intensity of flow cytometry is directly related to the concentration of cells. If the cell concentration in the sample is too low, the flow meter may not be able to detect enough cells correctly, resulting in a weak signal

5. Fluorescence channel and expression amount mismatch
Low antigen expression abundance should be selected for strong fluorescence channels, such as PE-CY7, etc., if selected APC-H7 may lead to signal

6. Abnormal Number of Samples Collected
1. Insufficient density of cells used, resulting in insufficient collection, and sample loss during washing, resulting in insufficient sample loading

2. The flow cytometer pipe is blocked, not cleaned in time, or the prepared sample is clogged again, causing the machine to be blocked. The sample can be filtered before loading to avoid clogging the machine. Some cell types may precipitate on the wall of the instrument during the flow of the flow cytometer, causing the flow rate to slow down or be blocked. This may be due to factors such as cell size, concentration, and surface properties

3. Before using the instrument, use QC samples to check the operation of the machine to ensure the normal use of the machine. Some use peristaltic pump injection machines. The peristaltic tube is aging and has problems such as aging for a long time

4. Improperly set flow rates and pressures may cause cells to aggregate, settle, or fail to fully pass through the flow cytometer or use too large a sample cell volume, causing the sample to be collected too quickly