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Signal not correctly compensated
Check positive single color control is set up correctly on flow cytometer, gated and compensated correctly to capture all the events.
Insufficient antibody present for detection
Increase amount/concentration of antibody.
Intracellular target not accessible
Check if target protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent internalisation of cell surface proteins, everything must be done on ice or at 4°C, with ice cold reagents, to stop all reactions.
Adding sodium azide will prevent the modulation and internalization of surface antigens which can produce a loss of fluorescence intensity. For staining of cell lines, trypsin can often induce internalisation of cell surface proteins, particularly cell surface molecules, and more gentle detachment methods may be required.
Intracellular staining – fluorochrome conjugate too large
Fluorochromes for intracellular staining experiments should have low molecular weight. Large molecular weight flourochromes can reduce antibody motility and possibly its entry into the cell.
Lasers not aligned
Ensure lasers on flow cytometer are aligned correctly by running flow check beads and adjusting alignment if necessary. If the lasers do not align correctly or if drift occurs, you may need to consider having the machine serviced.
Target protein not present/expressed at low level
Ensure tissue/cell type expresses target protein and that it is present in a high enough amount to detect.
Soluble/secreted target protein
Is the target protein soluble and secreted from the cell? It needs to be membrane bound or cytoplasmic to be detected easily by flow cytometry. A golgi-block step, such as with Brefaldin A, may improve the signal achieved for intracellular staining.
Offset too high/gain too low
Use the positive control to set up the flow cytometer correctly again, using the offset to ensure the fluorescent signal from cells is not being cut off, and increase the gain to increase the signal (within reason – care should be taken).
Fluorochrome fluorescence has faded
Antibody may have been kept for too long or left out in the light. Fresh antibody will be required.
The primary antibody and the secondary antibody are not compatible
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary).
Antibody concentration too high
This will give high, non-specific binding or very high intensity of fluorescence. Reduce the amount of antibody added to each sample.
Excess antibody trapped
This can be a particular problem in intracellular staining where large fluorochrome molecules on the antibody can be trapped. Ensure adequate washing steps and include tween or triton in wash buffers.
Add 1% to 3% blocking agent with antibody as well as a blocking step.
Gain set too high/offset too low
Use the positive control to set up the flow cytometer correctly again, using the offset to reduce background from small particles and reduce the gain to decrease the signal.
Decrease the antibody concentration. Detergent can also be added to the wash buffers to ensure any excess antibody is washed away.
More than one cell population present expressing target protein
Check expected expression levels from the cell types contained in the sample and ensure adequate cell separation if necessary.
Cell doublets present
Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot. Mix cells gently before staining and again before running on the cytometer using a pipette. Cells can also be sieved or filtered to remove clumps (30 μl Nylon Mesh).
Ensure cells in the sample have not lysed and broken up. Samples should be fresh and prepared correctly. Do not centrifuge cells at a high rotor speed or vortex too violently.
Ensure sample is not contaminated. Bacteria will auto fluoresce at low level. This will also give a high event rate.
Low number of cells/ml
Run 1x106 cells/ml. Ensure cells are mixed well (but gently).
Cells clumped, blocking tubing
Ensure a homologous single cell suspension by pipetting gently several times before staining. Ensure you mix again before running. In extreme cases, cells can be sieved or filtered to remove clumps (30 μl Nylon Mesh).
High number of cells
Dilute to between 1x105 and 1x106 cells/ml.