Fluorish Panel Design Tool

Introduction

The tool is aimed at making the development of new flow cytometry antibody panels easier for you, your core facility manager or tech, and other scientists who share your interests.
Here are a few of the highlights:

• Your instrument's configuration is used by Fluorish to effectively and accurately assign fluorescent proteins, nucleic acid dyes, lipid binding stains and any antibody bound fluorochromes to an appropriate channel.
• You have access to the catalog information from all vendors whose logos show on the left of this page, totalling over 32,000 antibodies.
• Assign and restrict fluorochromes to their optimal excitation and specific detection range.
• We assist in detecting possible cross beam compensation issues and providing for fine-tune controls from the user.
• Integrate with your custom inventory through Fluorish Labs.

Metadata and Logging In

Fluorish wants to make antibody panels easier to find and share, so we encourage entry of metadata that may help you or other users find your panel in the future. Logging into your Fluorish Lab makes it easy to utilize your lab's reagent inventory in the creation of your panel(s).
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• Start New Panel- this will clear all information from the design tool. All previous work saves automatically, so if a panel has been made, it is accessible when the program reopened. To clear it, click the start new panel button.
• Edit Saved Panel- this will launch a file finder window to locate existing panels. All of the information will load into all panes, including the final selections. This allows the user to go through and edit any information from an existing panel.
• Custom Antibody Database- the Fluorish database contains all antibody information from partner vendors. However, a number of users have their own custom conjugates created in the lab. There may also be a number of purchased antibodies in stock within the lab for which the user does not want to enter in the vendor information. Hence, the Custom Antibody Database or Lab Inventory Database tool at fluorish.com will provide an interface to enter in all relevant information regarding antibodies present in the lab stock. This will be accessible in the Panel Wizard and selections can be made from it as well.
• The Custom Antibody Database- is stored at fluorish.com and is shared with the rest of the lab. The user account on Fluorish.com will provide access privileges to the lab members or institution members.
• The Custom Antibody Database- will provide a single, unified source of information to store, edit and share all custom conjugated antibodies within the lab.
• View All Reagents- will take the user to the online database of all our partner vendors at Fluorish.com. Here the user can use specific keywords to restrict the search. There is also an option to build a panel manually if desired.
• Experimental Details- Use the text boxes to enter in relevant information. If an additional field is required, a button is provided in Pane 1 as well.

Instrument Configuration and Confirmation

You cytometer configuration is an integral part of antibody panel design. Fluorish allows you to input your custom configurations and uses its configuration to refine the antibodies that can be used in your panel(s).
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• Default Configurations- the default configurations for every cytometer are programmed into our database and available to use in your design.
• Build Your Custom Configuration- you can create files that contain your custom configured cytometers here. This tool will allow the you to enter in the laser wavelengths and channel detection ranges for the instrument. These files can be created and saved to your Fluorish Lab account, and then become available to any other user at your institution.
If you use BD FACS Diva software, the CS&T platform within Diva allows you export a file containing the instrument configuration that can then be dropped into the Fluorish panel design tool.
• Choose From Your Institutions Cytometers- if you're logged in, and you or others at your institution have uploaded custom configurations, they will be available to you to choose from.
• Assign a Dump Channel- choose a dump channel and the design tool will leave this channel open allowing the entry of multiple targets into that channel

Select Your Dyes

Fluorish considers unconjugated fluorescent probes to be a primary step in creating a panel. Typically a particular fluorescent protein (like GFP) or a particular DNA binding dye (like PI) is used as the centerpiece of panel construction.
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• Dye Assignment- based on your instrument, the design tool identifies and displays all fluorescent molecules that could be used in the panel. This is accomplished by cross referencing the excitation and emission wavelengths of every fluorescent molecule, with the specified instrument configuration. Any selection made at this step will automatically assign optimal detection channels.
• Missing Molecules- if a fluorescent molecule is missing from the list, we'd like to help so please let us know: office@fluorish.com
• Manual Channel Assignment- if the wizard makes a channel assignment that is suboptimal, you can open the configuration of the cytometer and make any manual channel assignments at this point (from Help).
• *Caution is advised in changing channels. An optimal channel is chosen based on excitation and emission profiles. Moving a fluorochrome from one channel to another and then filling the original channel with another fluorochrome can result in potentially significant compensation issues.

Choose Your Fluorochromes

Further narrowing of available reagents is based on the fluorochromes selected for your panel. The tool automatically selects the fluorochromes that match your instrument configuration and available channels.
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• Select From Available Fluorochromes- based on your instrument's configuration and the dyes previously selected, fluorochromes that can be used are selected with a checkmark by the tool. If a fluorochrome is not selected, it can either not be detected by any channel based on the instrument configuration or the optimal channel was filled by a fluorescent molecule during dye selection.
• Custom Options- click the help link to access your Fluorish Lab if a custom fluorochrome or modification is required. If needed, you can again access instrument configuration and make any manual channel assignments to close a specific channel.
*Manual assignments at this step may be desired if there is a particular antibody conjugate that is absolutely required.

Specify Your Targets

Species and targets are the last refinement step of the panel creation process in the Fluorish design tool.
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• Species- select a species from the species buttons at the top of the screen.
• Targets- begin typing in the Target List field and select look for your target in the Target List. Add as many targets as desired.
*Primary and alternate names are both considered during database selection
• Specific Clones- for each target added, specific clones can be set to only provide search results for that antibody clone.
• Custom Options- again, your custom antibody database can be accessed to add any additional targets of antibodies n the Fluorish provided database.

Make Reagent Selections

Based on the instrument configuration, dyes, fluorochromes, and targets you've selected, both your custom antibody inventory and the Fluorish database are queried for available reagents and the results are displayed for your choosing.
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• Choose by Target- the number of formats for each target are presented next to each target in parentheses in the Target list.
• View by Specific Vendor or Format- targets can be refined further by selecting one or more vendors or formats from the respective lists.
*Hold down the control key while clicking to make multiple selections.
• Select Your Reagent- double-click on a reagent in the main upper list and it will be assigned to the optimal channel of your instrument configuration. The channel will no longer be available and the results will refine based on this channel's "closure". If an antibody/fluorochrome conjugate is selected that can be excited and detected by detectors off different lasers, you'll get a dialog that will ask you to assign the channel based on excitation efficiency of the laser and the channel(s) appropriate for detection.
**If a fluorochrome was manually selected during fluorochrome selection because it is not ideally excited and detected by any laser/channel combination, and an antibody is selected with that particular fluorochrome conjugate, the tool will request that the user manually assign the appropriate detection channel.
• BIOTIN and PURE- options are available at the bottom of the format list. If a BIOTIN and/or PURE option is used, you'll be prompted to select whether or not a secondary fluorochrome tag will be used. If yes, you will be prompted to select the fluorochrome for the secondary tag. If no, no channel assignment is made.
The secondary tag dialog box includes every fluorochrome in our database. If the fluorochrome is black, this fluorochrome will fit the instrument configuration and if selected, the channel assignment will be made automatically. If the fluorochrome is grey, this indicates that it is not optimal for the instrument and will not be automatically assigned if selected. Grayed fluorochromes can still be selected, but manual assignment into a channel will be required. Two other tools are available from Pane 7 The Auto Fluorish button will allow the user to enter in the relative density of antigen and based on relative brightness of selected fluorochromes and channel availability, the Fluorish panel design tool will propose the ideal "suggested" panel. Refinements can be made in Pane 8, but this tool provides a starting point based solely on relative brightness of the fluorochromes and antigen density. It does not take into effect the cell type, detector sensitivity, instrumental setup, autofluorescence, antibody clone or a number of other factors that can effect stain index. The Grid Tool button will show the target listed in rows and each channel of the instrument as a column. The number of each format available for that target and its corresponding channel are shown in each cell of the grid. When a cell is closed, the channel and target are grayed, indicating a refinement. This tool can help better select combinations of targets and fluorochromes If any antibody from a manufacturer is missing from the list, please let us know immediately so we can refine our database (office@fluorish.com). Changes to the database can be made quickly so within hours, the refinement can be made and the database updated.

Summarize, Finalize, Upload and Share

When your selections are complete, view your summary and finalized panels and upload to share with your lab, insitution, or the Fluorish community, or print or save your panel.
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