Note: If you need help beyond our Panel Builder Tool to design complex panels for your experiments, we have a team of technical support scientists available to help you. We know that building a complex flow cytometry panel can take time and can seem overwhelming. While we can’t run the experiment for you, our team can help design your panel to minimize spillover and increase resolution. As you run the panel, we can provide further support to optimize the panel to give great results. Contact us for help by clicking the Build Panels with an Expert button above.
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.
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.
Watch the video to create a panel for your spectral instrument
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.
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.
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.
When you are done, click on the Next Step button.
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.
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.
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.
Fluorophores emit light with varying levels of brightness (Figure 2). When choosing fluorochromes on the panel builder, we suggest:
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
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:
R&D scientist Natalie Oxford share her learnings for a flow cytometry panel that can help you get published.
|Organic dyes—small, stable molecules||Original||FITC|
|Pacific dyes||Pacific Blue|
|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|
|Large, protein‐based molecules||Original||APC|
|PE–Alexa Fluor 610|
|PE–Alexa Fluor 700|
|APC–Alexa Fluor 750|
|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|
|Brilliant Ultraviolet dyes and their tandems||Brilliant Ultraviolet 737|
|Brilliant Ultraviolet 805|
|Specialty dyes||NovaFluor Dyes and their tandems||NovaFluor Blue 510|
|NovaFluor Blue 530|
|NovaFluor Blue 555|
|NovaFluor Blue 585|
|NovaFluor Blue 610 30S|
|NovaFluor Blue 610 70S|
|NovaFluor Blue 660 40S|
|NovaFluor Blue 660 120S|
|NovaFluor Yellow 610|
|NovaFluor Yellow 660|
|NovaFluor Yellow 690|
|NovaFluor Yellow 700|
|NovaFluor Yellow 730|
|NovaFluor Red 660|
|NovaFluor Red 685|
|NovaFluor Red 700|
|NovaFluor Red 710|
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.
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.
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.
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.
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.
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.
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.
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