Flow Cytometry Panel Builder

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.

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.

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 fluorophores with a low complexity score.

Darker colored boxes indicate how difficult unmixing will be. Lower score means easier and more accurate unmixing. Separation tends to be better with lower complexity score and flurophores will be easier to distinguish.

Select the products and packaging sizes that you want. If you didn’t specify a clone earlier, a variety of clones will be displayed.

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.

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