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Our Scientific Support flow cytometry experts have put together a top 10 list of things to consider to help produce successful results from your flow cytometry experiments. We hope you will find these useful.

1. Sample preparation

Generally, each sample should contain between 1 X 105 to 1 X 106 cells /ml in 100 µl in PBS containing 1% BSA or serum.

Keep the samples on ice at all times during preparation and staining, and at 4^oC for storage after staining. Ensure a single cell suspension to enable accurate data and to ensure the fluidics system does not block. Cell suspensions can be filtered to remove clumps, e.g. use a 50 µm Nylon Mesh, and vortex immediately before running on the flow cytometer.

Blood samples:

Use fresh samples collected in tubes containing anticoagulant and transferred to the laboratory on ice. Refer to the supplier regarding the type of collection tube and anticoagulant for certain experimental settings (EDTA, Citrate, Heparin).

There are several commercially available red blood cell lysis buffers, and we would recommend to follow the protocols provided by the manufacturer. Alternatively, red blood cells can be removed from the sample using the following commonly used red blood cell lysis buffer (prepare fresh):

• KHCO3 10 mM
• NH4Cl 150 mM
• EDTA (pH8) 0.1 mM

Tissue samples: Use enzymatic methods or a homogeniser to create a single cell suspension. Cell culture: Harvest cells when they are about 70 – 80% confluent to ensure they are viable. We recommend avoiding samples that are over confluent and/or may contain too many dead cells or debris. Do a cell count of the single cell suspension using trypan blue.

»view our cell count protocol

For all sample types, ensure cells are >95% viable before staining. This will help to ensure the results are accurate and that there is as little debris as possible.

2. Fixation

Depending on the sample type, sample may need to be fixed post staining.

Membrane proteins:

Samples are usually fixed after antibody staining with 0.1 to 4% PFA for 10 minutes to overnight. If they are not fixed, they will need to be run through the flow cytometer to collect data immediately after staining.

Intracellular proteins:

Permeabilization is required for intracellular staining, to allow access of the antibody into the cell, and the cells must be fixed before they are permeabilised. For sample, fix with 0.1% PFA for 10 minutes, then permeabilise with 0.1% Saponin for 10 minutes. Or when using acetone or methanol to fix (10 minutes ice cold), these solvents will both fix and permeabilise. (Note that acetone can sometimes increase autofluoresence and adjustment of settings may be required).

Note: Fixation can change the FCS/SSC light scatter properties of the cells

Health and safety: Unfixed cells may expose the operator to additional biological hazards and we would recommend to follow the correct health and safety guidelines for handling the samples. For more information on fixation and permeabilization view our Intracellular staining protocol.

3. Lasers alignment

Ensure lasers on the flow cytometer are aligned correctly by running flow check beads and/or single color control beads or samples, 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.

4. Use the correct controls to set up the instrument

Use the positive, negative (or isotype) controls to set up the flow cytometer correctly. Other controls required are listed on our 'recommended controls' flow cytometry webpage:

5. Event rate

The flow rate is usually set around 50 µl/min. Most flow cytometers have 'low', 'medium' and 'high' flow rate settings to select from. Some machines will have an upper limit. If the event rate is very high, even when the flow rate is low, try diluting the sample in some buffer. Try to standardize flow speed and event rate for each sample.

Data is more accurate if the sample speed is slightly slower Lower flow rate is used when greater resolution is required, e.g. for DNA analysis. The data will be more accurate if the samples speed is slightly slower.

Higher flow rate can be used for qualitative measurements e.g. immunophenotyping. In this case, data is less 'resolved/ as the cells are not so well aligned to the laser. But the data can be acquired more quickly.

6. Gating strategy

Gating allows the user to select regions of cells or a cell population. Gating is dependent on experimental settings and the information required from the data. New software now available allows for various gating shapes, and we would recommend to review the software instructions for further information on your machine.

Gating tips:

1. The first gate should always be set on the correct cell population in the SSC/FCS plot.
2. Regions drawn on negative populations (excluding scatter and viability gates) need to be pushed to the very edge of the x y axes to avoid excluding negative events that are crushed up against the axis.
3. Data follows a biological distribution that does not always fit with rigid gating. Be consistent and logical in how you draw regions, to minimize subjective bias for inter experiment comparison. We would recommend where possible to run all the samples from one set of experiments using the same machine settings and gates to ensure standardization.

7. Secreted proteins

Detection of secreted proteins is difficult as the protein will be released from the cell before detection, or may degrade rapidly. A frequently used solution is to block the secretion using a Golgi Block such as Brefeldin A and other compounds. Cells can incubated for 4 – 16 hours with Brefeldin A which prevents proteins being released from the golgi. The exact experimental conditions and time will need to be optimized. Any cells expressing the protein can then be detected. In this case, fix and permeabilization is required before staining to ensure the protein remains intracellular.

8. Multicolor analysis and compensation

When using multicolor analysis using different fluorochromes, emission spectra can overlap, fluorescence from more than one fluorochrome may be detected within one channel. Take care to select fluorochromes whose excitation and emission spectra overlap as little as possible. Check that the correct appropriate filters are selected and data is compensated for accurate results. This ensures that the fluorescence detected in a particular detector derives solely from the fluorochrome that is being measured. More information on compensation can be found on the following protocols page from our website:

• Fluorescence compensation in flow cytometry
• View our guide to fluorochomes, including a list of excitation and emission wavelengths.

9. High background

Sample line not clean

Flow cytometer settings not correct

Excess antibody

Dead cells and debris

Ensure cells are 95% viable before staining and that the sample does not contain too much debris. The threshold can be adjusted to remove debris from the histogram, but the threshold should not be set too high as the data will then be inaccurate. It is better to start again with fresh samples if possible, rather than use a sample containing a lot of debris.

10. No staining

Signal not correctly compensated

Check positive single color control is set up correctly on flow cytometer and gated/compensated correctly to capture all the events.

Insufficient antibody present for detection

Increase amount/concentration of antibody.

Intracellular target/epitope not accessible

Check if target protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent internalization of cell surface proteins, everything must be done on ice or at 4°C, with ice cold reagents, to stop all reactions. For staining of cell lines, trypsin can often induce internalization and degradation of cell surface proteins, and more gentle detachment methods may be required.

The antibodies to some membrane proteins may detect an epitope in the cytoplasmic region of the protein, in which case gentle permeablization 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. For example, tandem dyes will be larger.

Machine settings

Use the correct controls (see point 4) to ensure the machine is set correctly.

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.

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).