Introduction to Antigens

INTRODUCTION TO ANTIGENS

Now that we have named our experiment, specified a species, and selected our instrument, FluoroFinder has been provided with information about the configuration of our cytometer and can find relevant antibodies for our experiment. The FluoroFinder algorithm uses this information during panel design to determine what fluorophores can be detected by your cytometer, and in what channels. Having completed this, we are now presented with the next step in the process, Antigens.

In this step we will provide FluoroFinder with a bit more information about our experimental design, in particular the species we will be working in, what antigens we would like to detect, and (if provided) details about the expression pattern(s) of those antigens.

Navigating the Antigens Page

NAVIGATING THE ANTIGENS PAGE

To add antigens of interest to your panel, please follow these steps:

Enter Antigens - Antigens can be entered in multiple different ways:
- Antigen entry boxes - available for individual antigen entry. In these boxes, one can enter an antigen of interest (e.g. CD45). This entry will search the FluoroFinder database for antigens of the same name associated with the selected species and display them for selection
  
  In the example above, CD45 has been entered into the first of three antigen entry boxes. Once entered, FluoroFinder searches its database and loads relevant fluorophore conjugates for our experiment, represented by the colored dots to the right of the antigen name.
- Bulk Adding Antigens - Above the antigen entry boxes is a link with the text "Already have a list of antigens you want to use?"
  
  Selecting this option will open a dialog box for entry of lists of antigens. Values can be copied and pasted from excel, or entered one antigen per line, into this popup and the antigens will be searched, identified, and available for addition to the panel when selected.
Add Additional Reagents – After antigens have been selected, you may want to add additional fluorescent reagents (Viability stains, Functional Reagents, Fluorescent Proteins, Apoptosis Reagents, etc.) to your panel. Beneath the header labeled “Add Additional Reagents” is a search function that allows you to directly search for additional reagents by name, or to specify a category of reagent and search within that category. To use this section:
1. (Optionally) Select a category to search from the drop down menu:
2. Type your search criteria in to the search box:
3. Select the reagent you'd like to add to your experiment. It will be added in a new row to indicate your selection:

3. Continue – once you’ve selected your antigens, you may want to configure additional options prior to continuing ((see “Antigens: Options”)). If so, configure them now. Once finished, click “Continue>” at the top of the page to move to “Products”

Antigens: Additional Options and Configurations

ANTIGENS: ADDITIONAL OPTIONS & CONFIGURATIONS

There are a variety of options available within Panel Builder to customize your entry, 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.). For more information on Intellipanel, please contact support@fluorofinder.com

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 “Products” 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

Template Panels

Template Panels – Selecting “Template Panels” will allow you to select a previously saved panel (user saved, lab saved, or OMIP) to use as a template for your new panel design. Settings from the previously created panel (antigens, fluorophores, products, etc.) can be imported to your new panel to ease the antigen entry process

Expanded Antigen Options

Expanded Antigen Options – Next to each entered antigen, an options menu will appear (closed by default) that allows for configuration of additional details for each antigen. Clicking the “three lines” icon: next to each antigen will expand the options menu for that antigen. Expanded options for each antigen include:

Antigen Density

Antigen Density – the Antigen Density slider option provided FluoroFinder with information about the expected expression level of your antigen on your target cells. When set to “Any” (by default), FluoroFinder will not consider antigen density in its recommendations. Selecting other options will affect recommended fluorophore options as follows:

Antigen Density - Low - Antigens indicated as “Density – Low” will be matched with bright fluorophores to ensure they are visible
Antigen Density – Mid – Antigens indicated as “Density – Mid” will be matched with medium brightness fluorophores, avoiding insufficient signal or signal saturation
Antigen Density – High - Antigens indicated as “Density – High” will be matched with dim fluorophores, as those should be sufficient to produce a viable signal

NOTE: Providing accurate antigen density information is crucial to the performance of the Intellipanel feature.

Co-Expression

Co-Expression – Co-Expression allows for the creation of groups within the panel design process. These groups are meant to contain markers that are known to be “co-expressed” on the same cell type (e.g. CD45, CD3, and CD4 are all expressed on Helper T-cells, and may be placed in a Co-Expression group called “Helper T-cells”). Creating Co-Expression groups that accurately inform FluoroFinder about the expression patterns of your antigens can help in the organization of the panel design process.

NOTE: Providing accurate Co-Expression information is crucial to the performance of the Intellipanel feature. Intellipanel will attempt to minimize spectral overlap between antigens in a Co-Expression group. This will result in lower Complexity Indices for Co-Expression groups, and likely ease the process of identifying expression patterns in the resulting panels.

Target Species

Target Species – Under Target Species, you can select an individual “Target Species” (aka Isotype) for each of your antigens. This can be particularly useful if working with samples from a transgenic model

Clone

Clone – if you know that a specific clone of antibody works best for your application, you can limit the selections that FluoroFinder will make for that antigen to ONLY antibodies of that clone. NOTE: restricting your search by Clone may result in quality products being eliminated from recommendations down the line. It is recommended that you filter by Clone, if desired, during the Product Selection step.

Fluorophore

Fluorophore – if you know that you would like to use a specific antigen/fluorophore conjugate in your panel, you can limit the selections that FluoroFinder will make for that antigen to ONLY antibodies conjugated to that fluorophore. NOTE: restricting your search by Fluorophore may result in quality products being eliminated from recommendations down the line. It is recommended that you filter by Fluorophore, if desired, during the Product Selection step.

In Dump Channel

In Dump Channel – often, particularly in high parameter panels, users would like to include markers/cells to be eliminated from analysis in a single “Dump Channel”. Frequently, this channel will be shared with a Viability marker to exclude dead cells (along with the other cells to be eliminated from analysis). In this manner, a single fluorescent channel can be used to ensure many cell types are removed from the analyzed population. Enabling this slider for an antigen (or antigens) will allow for the antigen(s) to be assigned to a single fluorescent channel.