Multicolor flow cytometry with 5 easy steps
With this tool, you can:
- Create a new immunophenotyping experiment or add antibodies and reagents to an older panel
- Insert antibodies already used in your experiment
- View fluorochrome spectrums to quantify emission spectral overlap
- Export an excel document with your antibody choices or directly order
Getting started with the flow cytometry panel builder tool
Choosing the optimal combination of fluorochromes can be simplified with a guided method. The Invitrogen Flow Panel Builder offers a customizable panel building process to fit your flow cytometry experimental needs, whatever your experience level.
5 step video Staining indexes Immune cell markers Relative antigen density Tips and tricks videos
Video: how to use the panel builder
Watch the video to learn how to use the Invitrogen Flow Cytometry Panel Builder to build your next flow cytometry panel in 5 easy steps.
Hi, my name is Nathalie, and I am an R&D scientist with Thermo Fisher Scientific. I will present a macrophage panel design with the Invitrogen flow cytometry panel builder.
Step 1
The first step is picking our instrument with the appropriate laser and channel configuration. Note: there is an extensive list of instrument options. Filter by typing in your cytometer name. Edit your settings, such as the band pass filters and wavelength, or simply build your cytometer from scratch using your own parameter configurations.
Step 2
Our next step is to enter our markers and viability dye. For this experiment, the target species is human. Enter any antigens that you currently have on hand. Enter any fluorescent proteins, such as GFP. And next, enter the markers you need to pick fluorochromes, or purchase, or try.
You can enter protein abundance because that will help with picking the correct fluorochrome in step 3. If you don’t know, put the bar to medium. Example: CD14 and CD68 is high expression of protein.
Another great feature is our dump channel option. Selecting a dump channel will give those markers the same fluorochrome, and it is usually reserved for the negative or unwanted markers. Example: we have CD56, CD45R, and CD3 in the dump channel which respectively are our natural killer, B, and T cell markers. To set your target into the dump channel, you go to edit more options, and select a yes or no.
Remember to add your non-antibody reagents, including a fixed viability dye.
Step 3
Step 3 is picking the fluorochrome for each specific anybody.
As a rule of thumb, low expressing targets should use brighter fluorochromes while high expressing targets use dimmer fluorochromes. Brighter is not necessarily better, as that can result in spreading error from one channel to the next. In step 2, if you indicated the protein abundance, the webtool can suggest fluorochromes with a black flag.
Example: if we go back to the protein abundance CD60, we indicated the target to be expressed highly. The webtool recommendations are indicated by the black flags. As you can see here, with the CD60 target, we set high protein abundance back in the second step of the webtool panel builder. So, the panel builder in the third step (here) recommends FITC, which is a dim format.
Each of these dots represents an available for chrome for this target. There are six available colors in the row for CD14, and some are recommended.
There is a built-in SpectraViewer to look at the selected fluorochrome emission spillover and spread. This populates as you pick your colors. You can click on the SpectraViewer on top, and it shows up full screen. The SpectraViewer is adjusted by each individual laser.
Example: If we deselect the fluorochrome choice for CD68, the emission in the SpectraViewer disappears.
Step 4
Let’s look at clone selection. Some targets will have multiple clones available. If you click on the figures for the clones, additional information pops up. Some of the clones will have undergone advanced verification, meaning the antibody binds to the stated antigen.
Step 5
Here, you can save your panel or purchase. And with this, we are one step closer to beginning our experiment.
5 steps of the flow cytometry panel builder
Following 5 simple steps, anyone can design a flow cytometry panel. Whether they are new to flow cytometry or experts in the field, the panel builder can help.
Cytometer configuration
Knowing your cytometer’s configuration is the first step in panel design. Use the drop-down menu containing over 70 instruments to select your cytometer.
If the bandpass filters shown do not match what is in the instrument you will use, simply click the “edit cytometer settings” to adjust the lasers, channels, or bandpass filters as necessary.
If your cytometer is not found, simply enter the configuration manually. Select your lasers and set up the channels and filters as necessary for your cytometer.
Antigen selection
Are you designing this panel with a key antibody conjugate that you already have in your lab and will not need to purchase? If so, click yes and enter the antigen name, fluorochrome, and intended channel. Then click “add to panel.”
Now begin adding the other antigens by first selecting your target species from the drop-down menu. Type in the antigens you need, clicking “add another antigen” until all of your antigens are listed.
Select Protein abundance, and move the bar from high to low. If are unsure what it should be, leave it at the midway point. Adding this will help the panel builder program suggest fluorochromes in step 3.
If you would like to specify a specific clone, simply use the advanced options to select the desired clone.
Want to assign something to a dump channel? Open the advanced options for the antigen and move the slider to the right, to select yes. This will allow you to assign more than one antigen to this channel.
Don’t forget the viability dye! Select either the fixable or non-fixable option.
When you are done, click on the Next Step button.
Fluorochrome selection
Fluorochrome selection is guided by the spectral information of each fluorochrome. This step allows you to visualize the spectra of selected fluorochromes across the top of the page as you select fluorochromes for each antigen shown on the left.
Before any selection is made, the number of available channels for each antibody is shown. With each antigen selection, another spectrum will show at the top of the page. A matrix view allows you to see the antigens listed in the rows and with the channels shown in the columns.
A black flag in the corner indicates the recommended fluorochrome choice based on protein abundance from step 2.
Begin making fluorochrome selections. Start with the top row which lists the antigens with the fewest fluorochrome options available. Work down the table to the antigens with the most fluorophore options on the bottom. To guide you in fluorochrome selection, spectral information is provided. In general, the least spectral overlap is best.
Continue selecting your options to match all antigens with a fluorochrome. When done, check that your choices will work for your instrument by clicking on the SpectraViewer button.
The full spectra of all fluorochromes per laser will show and be labeled. View the estimated light captured by the bandpass filters in your instrument. Be sure to check the theoretical spillover values for issues.
If the fluorochrome you want to select is not listed, simply click to add a placeholder fluorochrome.
Enter your fluorochrome, click add and select to add it to your panel.
Placeholder fluorochromes can be used at any time, even if no other fluorochromes are listed. Simply click on the plus sign, and then enter and select your fluorochrome of choice. When you click on the SpectraViewer link you will be able to visualize your selected fluorochrome.
Product selection
Select the products and packaging sizes that you want. If you didn’t specify a clone earlier, a variety of clones will be displayed.
Need additional information to help you decide? Click on an image in the product selection listing to enlarge it. Clicking on the product name will open the product data page in a separate window.
Panel summary
After completing product selection, step 5 allows you to review all of your selected antibodies per laser type and the resulting excitation spectra to be captured by the filter sets of your cytometer.
Want to review the spillover matrix? Simply click the SpectraViewer button to see the matrix.
Not satisfied? Want to change something? You can return to editing by clicking the “Edit panel” button. Alternatively, you can click on the progress bar at the bottom of the screen to move back to a different step. At any time in the process, once a step has been completed, you may click on a step in the progress bar to return to it.
Once you have reviewed and are satisfied, you are almost done. Be sure to save your flow cytometry panel and give it a name. There are also options to export the panel design data as a spreadsheet or to download a PDF for printing.
Lastly, simply add the flow cytometry panel that you designed to your cart for purchase. If you are outside the US, the pricing in the Panel Builder will show in your country’s currency, and when you click the “add all to cart” button, the correct pricing and currency for your country will also show in the cart summary field.
Invitrogen Flow Cytometry Panel Builder FAQs
- Can I go back a step without losing the work I did?
- Yes, the previous work is saved.
- My marker isn’t showing up under step 2 of antigen, but I see it in the catalogue. How can I remedy this?
- Select your target species first, and then type your antigen.
- How do I share my panel with my lab manager?
- Export the spreadsheet found on step 5.
- What do the colorful dots indicate in the “Fluorochromes” step?
- The dots indicate the number of fluorochromes that Thermo Fisher Scientific has available in each channel on the cytometer you have chosen.
- My cytometer does not use a standard configuration. How can I make changes to one in the dropdown?
- Select a cytometer from the dropdown menu and then select the “Edit cytometer settings” link to modify the configuration as needed.
- I can’t find my cytometer in the dropdown. Can I build one?
- Yes, you can define a new cytometer using the “Enter your cytometer manually” link below the cytometer dropdown menu.
- I do not order products through thermofisher.com. What other endpoints are available to me?
- There are both PDF (printable) and CSV (spreadsheet) exports available in the summary step for you to take away for reference and ordering purposes.
- I plan to use antibodies that are not from Thermo Fisher Scientific. How can this Panel Builder be useful to me?
- Step 2 allows for antibodies you already purchase or from outside Thermo Fisher Scientific. If you are undecided about which format you want for something, you can add the antigen name in the “Antibodies you need” section and then choose a placeholder fluorochrome in the following step. Both will help you keep track of channels that are reserved for reagents that you do not plan to buy from Thermo Fisher Scientific.
Tips & tricks for building your flow cytometry experiment
Tips for picking fluorochromes
Fluorophores emit light with varying levels of brightness (Figure 2). When choosing fluorochromes on the panel builder, we suggest:
- Use a staining index for your specific flow cytometer. A staining index can be provided as a chart or table from a manufacturer who tested the same clone with different conjugates on their instrument. It is also possible to create a staining index with flow capture beads. For example, Figure 2 shows a staining index of the most popular fluorochromes analyzed on the Attune NxT Flow Cytometer. Each flow cytometer has different lasers, filters, and detectors that can influence the signal observed from a marker conjugated to a fluorochrome.
- Chose a combination of fluorochrome brightness based on your expressed protein levels. Using only very bright fluorochromes may result in spillover and can dampen the signal of a marker.
- Examine the mean fluorescence intensity for each fluorochrome and antibody clone to compare brightness. These can be found in product data sheets.
Want to know more about a staining index? Need the staining index for the BD LSR II Flow Cytometer? Find it in our Flow Cytometry Panel Design: Basics
Figure 2. Average stain index of fluorochrome conjugated to CD4 and analyzed on the Attune NxT Flow Cytometer.
Comprehensive list of available fluorophores based on their usage, benefits, and intended applications.
| Family | Type | Benefit | Invitrogen fluorophore |
|---|---|---|---|
| Organic dyes—small, stable molecules | Original |
| FITC |
| Pacific dyes |
| Pacific Blue | |
| Pacific Orange | |||
| Alexa Fluor dyes |
| Alexa Fluor 405 | |
| Alexa Fluor 488 | |||
| Alexa Fluor 532 | |||
| Alexa Fluor 561 | |||
| Alexa Fluor 647 | |||
| Alexa Fluor 660 | |||
| Alexa Fluor 700 | |||
| eFluor organic dye |
| eFluor 450 | |
| eFluor 506 | |||
| eFluor 660 | |||
| Large, protein‐based molecules | Original |
| APC |
| PE | |||
| PerCP | |||
| Tandem dyes |
| APC-Cyanine5 | |
| APC-Cyanine7 | |||
| PE-Cyanine5 (TRI-COLOR) | |||
| PE-Cyanine5.5 | |||
| PE-Cyanine7 | |||
| PE–Texas Red | |||
| PerCP-Cyanine5.5 | |||
| PE–Alexa Fluor 610 | |||
| PE–Alexa Fluor 700 | |||
| APC–Alexa Fluor 750 | |||
| PE–eFluor 610 | |||
| PerCP–eFluor 710 | |||
| APC–eFluor 780 | |||
| Polymer dyes—recent dye innovation | Super Bright dyes and their tandems |
| Super Bright 436 |
| Super Bright 600 | |||
| Super Bright 645 | |||
| Super Bright 702 | |||
| Super Bright 780 |
Tips for antigen density or protein abundance
Knowing your protein abundance will help you determine the best fluorochrome. Very bright fluorochromes are best used for low-abundance targets. Dimmer fluorochromes are fine for use with high-abundance targets. We advise:
- Gene expression levels may vary depending on cell source, stimulation, and protocols used for fixation.
- Dim fluorochromes to detect highly expressed proteins and bright fluorochromes to detect less-abundant proteins.
- Use flow cytometry compensation beads to help determine the specific density of your antigen. Internet-accessible databases can also help to find RNA expression levels.
Quick tips and tricks for your best panel
R&D scientist Natalie Oxford share her learnings for a flow cytometry panel that can help you get published.
Tip 1: Minimize spill-over by using the correct and separate channel
Any time you have markers that you know will be co-expressed on your cells of interest, make sure to space them out into separate channels. If you will need to use any adjacent channels, that's where you would put any markers that are mutually exclusive so that they'll still be easy to distinguish.
Tip 2: Intracellular targets need special buffer for fixation and permeabilization for staining
You'll also want to keep in mind the buffer that you're using to fix and permeabilize your cells, as we have several options. When you're looking at cytoplasmic targets, what the buffer is appropriate may not be the same as when you're looking at nuclear targets, because you want to make sure that you still have access to your antigens without over-fixing your epitopes.
Tip 3: Viability dyes are required to find live cells
A third tip I wanted to share with you is to always include a viability dye in your staining panel. This will help eliminate any false positives that are caused by dead cells or debris, because those can be sticky. You have a lot of options for choosing a viability dye, so you don't need to design your panel around them. You can build out the rest of your panel and optimize your core markers, and then fit in a viability dye in an empty channel.
Tip 4: Save your bright fluorochromes for dim targets
As you're building out your basic panel and you want to incorporate some more antigens, make sure you're keeping the density of your antigen expression in mind. So if you have antigens with low or unknown expression, those would be ones that you want to assign to your brightest dyes, such as PE or APC.
Tip 5: Try to combine negative markers in one channel (dump channel) to save space on your panel
A helpful trick when you want to exclude a lot of cell types at once without having to suck up multiple channels for that would be to use a dump channel. This is where you're placing all the antibodies that identify your cells that are not of interest into the same channel with the same fluorochrome, and then those can be easily gated out and all of the cells negative for the dump channel would be those that you use for your analysis going forward.
Immunology at work
Learn immune cell type markers and protocols. The Invitrogen Immunology at Work Resource Center is a learning center with technical content designed for new and experienced life scientists alike exploring the field of immunology.
Looking for quick tips and tricks? Find it in our Flow Cytometry Panel Design: Basics
Resources
Fluorophores excited by laser
Related products
Flow cytometers
Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.
Brilliant Violet and PE CF dyes are subject to proprietary rights of Becton, Dickinson and Company.
Cy™ is a trademark of Amersham Biosciences Corp. Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use.
For Research Use Only. Not for use in diagnostic procedures.
