Flow Cytometry Support—Troubleshooting

If the issue is clearly an instrument problem, send an email to Instrument Services at instrumentservices@thermofisher.com with your instrument serial number. If the issue is software or application-related, or if you do not know the source of the problem, send an email to Cellular Analysis Technical Support at technical-molecularprobes@thermofisher.com, including the words “flow” or “Attune” in the subject line for proper routing of your email.

Send an email to Cellular Analysis Technical Support at technical-molecularprobes@thermofisher.com, including the words “flow” or “Attune” in the subject line for proper routing of your email. Please describe the type of application you are doing, how you set up the experiment, and your results. It would be ideal if you could send us a sample of your data that we can review.

We recommend looking inside the box, possibly underneath the bag of buffer.

Open the Attune™ NxT software, open the main menu, and go to the "Instrument" tab. The option on the far left is "System Log". Click on that and it will open the log file. At the bottom of the page there is a button labeled "Export". Use this to export and save the log file.

There are several things that can cause a high event rate:

• There may be an air bubble in the flow cell
• The threshold level may be set too low
• The PMT voltage for the threshold parameter may be set too high
• The sample may be too concentrated
• The sample flow rate may be too high
• The sample may have a bacterial contamination

There are several things that can cause a low event rate:

• The system may be clogged
• The threshold level may be set too high
• The PMT voltage for the threshold parameter may be set too low
• The sample may be too dilute or not adequately mixed
• The sample syringe may be loose

There are three ways that our antibodies are classified to meet FDA regulations:

• In Vitro Diagnostic (IVD) products are validated for clinical use. They come with a specific protocol that must be used to obtain validated results. 
• ASR products have been validated to bind to a specific target, but do not have a validated protocol. In a clinical setting, the user must validate their own protocol in order to use these antibodies. We are not allowed to provide protocol information for these products. 
• Research Use Only (RUO) products are for the general user and are not validated for use in a clinical setting.

Antibody concentrations are sometimes posted on the website or printed on the label. When it is not there, look for a Certificate of Analysis (COA). COAs can be found at the bottom of the product page, just above the references. If it is not on the COA or there is no COA, send an email to Cellular Analysis Technical Support at technical-molecularprobes@thermofisher.com.

The best place to identify antibodies that will bind your cell type is in the professional literature. See what your colleagues are using and then call and find out if we have those antibodies.

Your cells should be in approximately 100 µL volume. One way to do this is to aliquot the correct number of cells (10E6) and then spin them down and decant the supernatant. The residual volume should be approximately 100 µL. 

For compensation, you need to prepare a singly stained sample (or compensation beads) for each color parameter that you are using. In addition, we recommend that you use FMO (flow minus one) controls. These are controls in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color. These controls are important for helping you properly set gates on your data.

No. Many users are using unstained cells in combination with FMO controls to identify their positive populations.

By running single-color controls, it is possible to remove signal of one fluorophore that spills over into the collection channel for another fluorophore. 

It is possible. But if you have a lot of values over 100, you probably need to look at the voltages that you are using for your data collection.

The first thing to do is check your threshold and see if it is set on forward scatter. If so, the beads are probably being excluded by the threshold. Reducing the threshold setting should reveal your beads. 

The formula has you divide the number of cells in the region you want to count by the number of beads analyzed. This value is then multiplied by the number of beads you added. The protocol calls for adding 50 µL of beads, and the vial of beads lists the number of beads per 50 µL— this is the number that you multiply by (do not divide by 50.) Finally, you divide by 1,000 to get your result as the number of cells/µL.

No, the data are not ideal when cells have beads attached to them. You need to use one of the Dynabeads™ Assays that allow you to remove the beads from your cells for downstream analysis. These include the Dynabeads™ FlowComp™ assays and DETACHaBEAD™ assays.

For help with designing panels for flow cytometry, see our new Molecular Probes Flow Cytometry Panel Design Tool. The panel design tool can help you choose fluorescent antibody conjugates for your flow cytometry panel in a few easy steps: pick the antibody species reactivity, select up to 14 targets of interest (choices include viability dyes), and choose the lasers or fluorophores you want to view.

Treating cells with trypsin or other reagents to detach adherent cells causes damage to the membrane, such that cells will be labeled with annexin V. The best way to avoid this problem is to allow your cells to recover for 30-45 min in the incubator. Swirl the tube/plate/flask every few minutes to prevent re-attachment. After this recovery period, you can label your cells with annexin V and analyze by flow cytometry.

There are several factors that contribute to the quality of the cell cycle profile. Cell number, dye concentration, incubation temperature, incubation time, flow rate (on a traditional flow cytometer utilizing hydrodynamic focusing), total number of cells acquired, elimination of dead cells, and removal of aggregates from data analysis should all be considered when analyzing the cell cycle.

The key to good generational profiles with CellTrace™ reagents is starting with cells that are evenly labeled so that they have a tight coefficient of variance (CV) when run at time zero after labeling. If the peak is too broad, the generations will overlap each other and the series of peaks will become a hump. Even labeling can be achieved by starting with a uniform cell population (not a mixture of lymphocytes and granulocytes for example) as staining will be proportional to cell size. Cells are labeled rapidly, so you want to pre-dilute the dye and mix it into your cells rapidly. Be sure that the cells are not sitting in a clump in the bottom of your tube. The easiest way to do this is to make a 2x dye solution (1x = 1–10 µM) and resuspend your cells in a half volume of medium (no serum or BSA). Add the dye to the cells and invert a few times to mix. Gently agitate the cells during staining. Once the dye incubation is over (20 min, 37°C), add serum or BSA (at least 1%) to scavenge any remaining unreacted dye. Spin down cells, wash 1x, and resuspend in complete medium. After a 10–20 min incubation to undergo de-esterification, cells are ready to be set up for whatever treatment you are planning. Be sure to keep a time zero control as you need to know where the first generation ran.

Many antibodies and stains will label dead cells. This will give you misleading data if you do not exclude the dead cells from your analysis. Of course, if you are labeling fixed cells, they are already dead and you do not need a viability stain. However, if you label your cells prior to fixation, then you need to use one of the LIVE/DEAD™ Fixable Dead Cell Stains. 

Indo-1, AM is the preferred stain for flow cytometry, where it is more practical to use a single laser for excitation, usually the 351–364 nm spectral lines of the argon-ion laser, and monitor two emissions. The emission maximum of Indo-1 shifts from ~475 nm in Ca²⁺-free medium to ~400 nm when the dye is saturated with Ca²⁺. Indo-1, AM is particularly suited for multicolor fluorescence applications.

ROS activity can only be measured in live cells. Fixed cells are no longer alive.

This will not work well because the dyes in this kit will also stain mammalian cells. You can probably distinguish cells based on their scatter properties, but the stain will not confirm your selection.

You can stain bacteria with a general stain such as BacLight™ Green Bacterial Stain (Cat. No. B35000) or BacLight™ Red Bacterial Stain (Cat. No. B35001). You can look at gram character (Cat. No. L7005), cell viability (Cat. Nos. L7007, L7012, and L13152), cell count (Cat. Nos. L34856 and B7277), and cell vitality. Cell vitality can be measured by membrane potential (Cat. No. B34950) or by metabolism (Cat. Nos. B34954 and B34956).

Electronic noise is due to stray light collected by the system optics and/or low-level electronic signals. Noise may be eliminated or reduced through correct setup of instrument threshold and photomultiplier tube (PMT) voltage. The easiest way to differentiate a bacterial population from other small particles and electronic noise is to label the bacteria with a fluorescent stain and use the fluorescence emission to identify the population of interest. SYTO™ 9 is a great stain to label all cells and is detected in the FITC channel.