Introduction to Products

INTRODUCTION TO ANTIGENS

With the information about our cytometer selection from earlier, and the details we provided about the antigens and fluorescent reagents included in our panel design, FluoroFinder has now compiled selections of commercially available products and paired them with the appropriate channels on our cytometer.

The next step for you is to select fluorophore conjugates for each of your antigens, and then finally choose products from FluoroFinder’s database that match those selections.

Navigating the Products Page

NAVIGATING THE PRODUCTS PAGE

To select fluorophores and add products of interest to your panel, please follow these steps:

Scroll down to the Marker vs. Channel display – scroll down to beneath the spectral viewer (empty when first loaded as no fluorophores/products have been selected) where you will find a grid displaying your selected antigens (markers) from the prior step against the available fluorescent channels on your cytometer. In the intersecting cells, available fluorophore conjugates for a marker will be shown in their detection channel. See the image below for an example:
In the above image, the red rectangle highlights fluorophores that are detected in the 355nm lasers 379/28 bandpass filter which are available conjugated to the CD45 marker. The number of dots (5) represents the number of available conjugates in the channel.
Hovering over the dots will load a pop-up which indicates which fluorophores are available in that channel

In the above image, we can see the pop-up containing the available fluorophores in the 355nm 379/28nm bandpass filter for CD45
Select fluorophores for each of your previously entered antigens/fluorescent reagents - Using the table outlined above, navigate through each of your entered antigens and select an available channel containing a fluorophore conjugate you'd like to use. Selecting an channel for an antigen will load a selection screen displaying all available fluorophores from your included suppliers (all suppliers by default) that are available in that channel, with details of filter coverage, warnings against spectral overlap, etc. Select a fluorophore to add to your panel:

When a fluorophore is selected for an antigen (Spark UV 387 in this case), a screen will pop up displaying the associated products found in FluoroFinder’s database. Select a product to “+Add” to finalize your choice.

Once a fluorophore/product is selected, the grid display will grey out the associated row and column to prevent any further selections for that antigen AND for the channel associated with the selected fluorophore. This is to prevent multiple antigens from being selected in the same channel. If multiple antigens are to be detected in the same channel, a dump channel will need to be created for this purpose (see: Dump Channels). When selecting antigens, be sure to use good panel design practices: where possible eliminate spectral overlap, separate antibodies for antigens expressed on the same cells across lasers, assign bright fluorophores to lowly expressed antigens, dim fluorophores to highly expressed antigens, etc.

Where Spark UV 387 is selected for CD45, fluorophores are available for selection in either the CD45 column or in the detection channel for the 355nm 379/28nm channel. All options are greyed out to prevent multiple antibody/fluorophore conjugates from being selected in the same channel.

As fluorophores are selected the Spectra Viewer at the top of the page will update with the excitation and emission spectra of your selections. These can be viewed by laser line, or all together, using the buttons at the top left of the viewer. Additionally, as fluorophores are selected, the Complexity Score, providing a measurement of the complexity of compensating your panel, will be dynamically updated to provide an overview of how your selected fluorophores may affect data spread (see: Complexity Score)

Spectra Viewer displaying 355nm laser and excitation/emission for selected fluorophore Spark UV 387. Beneath the Spectra Viewer is a dynamically updated Complexity Score, providing a measurement of how the selected fluorophores may affect data spread

Continue selecting fluorophores/products until you have made selections for each of the included antigens/fluorescent reagents you added to your panel .
1. Continue - once you’ve selected all your fluorophores/products, click “Continue>” at the top of the page to move to “Summary”
  NOTE: There are a variety of optional features that can help you to narrow product offerings, view antibodies already in your inventory, customize data displays within the “Select Products” page. See “Select Products: Additional Options and Configurations” below for detail on these optional configurations.

Select Products: Additional Options and Configurations

There are a variety of options available within Panel Builder to customize your search parameters and settings. Find instructions for their use below:

Intellipanel

Intellipanel – Turning on the Intellipanel slider will enable the automated panel design features of FluoroFinder (if you have an Academic Plus subscription or an Industry Subscription account). Intellipanel assists users by making informed product decisions for panel design based on user input, machine learning, fluorophore curation, etc.). NOTE: Enabling Intellipanel after fluorophores have been selected will affect the ability of Intellipanel to make recommendations. For best results, enable Intellipanel during Antigen Selection, or from being your design from the Intellipanel start page. For more information on Intellipanel, please contact support@fluorofinder.com.

SpectraViewer

SpectraViewer – By default, the slider for “SpectraViewer” is in the on position. Toggling the slider will turn off the SpectraViewer display. Within the SpectraViewer, users can further customize their display by choosing to display individual laser lines or all laser lines , and by selecting a light or dark theme. The options menu provides methods for export of the SpectraViewer display.

Suppliers

Suppliers – Selecting “Suppliers” will provide you with the option to filter recommended products based on the manufacturer (e.g. Miltenyi Biotec, Biolegend, etc.). ONLY products from your “Included Suppliers” selected will be displayed as results in “3. Product Selection” of the panel building process. Any manufacturer deselected (unchecked) will not be included in the search. It is recommended that you DO NOT filter by supplier at this stage to ensure optimal products are found for panel design.

Spillover Matrix

Spillover Matrix - Selecting "Spillover Matrix" will open the collapsed Spillover Matrix showing each selected fluorophore, its filter coverage in its own primary detection filter, and any filter spillover to other channels in which a fluorophore has been selected. This is a useful tool for determining how selected fluorophores may be contributing to spectral overlap and panel complexity.

Unexpanded Spillover Matrix Button

Expanded Spillover Matrix for Spark UV 387 and Brilliant Violet 510 on an LSR Fortessa X-20

Marker vs Channel - Data Display and Organization Options

Data organization options – FluoroFinder provides a variety of options on how to organize and display the data in the fluorophore/product selection table. Each of these options can be found just above the table in a linear display

. A description for each of these options is below:

Highlight Products in Inventory

- checking this box will place a message/indicator next to products that are in your inventory

Organize antigens by Co-Expression Group

- checking this box will change the organization of your antigens on the x-axis of the table, organizing them by any co-expression groups defined during the “Select Antigens” stage. See: Co-Expression Groups

Organize antigens by conjugate availability

- checking this box will change the organization of your antigens on the x-axis of the table, organizing them from that with the least available fluorophore conjugates to that with the most. This can be useful in identifying which antigens may be limiting factors in your panel design due to limited product availability

Adjust spillover threshold warning

- adjusting this slider will change the value at which FluoroFinder provides warning about spillover of a fluorophore into other used channels. FluoroFinder defaults spillover warnings to 50% filter coverage.