TITLE: LEGENDplex™ Assay Setup Protocol for Cytek® Aurora and Northern Lights Flow Cytometers
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>To view images that accompany this protocol, click </span><a href="https://www.biolegend.com/Files/Images/BioLegend/legendplex/instructions/Setup_Procedure_for_Cytek_Aurora_and_Northern_Lights_2_Lasers.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></div><div class = "text-block">This protocol provides step-by-step instructions for the setup of Cytek® Aurora and Northern Lights flow cytometers for data acquisition of BioLegend’s LEGENDplex™ multiplex immunoassays. </div><div class = "text-block"><span>A pre-made LEGENDplex™ Experiment Template that can easily be imported into the SpectroFlo® software is available for download at </span><a href="https://www.biolegend.com/en-us/legendplex" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">biolegend.com/en-us/legendplex</span></a><span> under the Instrument Setup tab. </span></div><div class = "text-block">If desired, users can also create their own Experiment Template by following the instructions found at the end of this protocol. </div><div class = "text-block">Unmixing of spectral data is not required. Please export Raw FCS files for downstream analysis. This is analogous to using uncompensated data on a conventional flow cytometer. </div><div class = "text-block">When analyzing LEGENDplex™ Assay FCS files generated on an Aurora or Northern Lights flow cytometer, the assay beads emission spectra should be analyzed using the following recommended channels: </div><div class = "text-block">• Without a yellow-green (YG) laser: Bead Classification = R4, Reporter = B4 </div><div class = "text-block">• With a yellow-green (YG) laser: Bead Classification = R4, Reporter = YG1</div></div>

[STEPS]
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Perform instrument start up procedure and instrument QC per manufacturer’s recommendations.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Import the LEGENDplex™ Experiment Template into the SpectroFlo® software, or follow the instructions at the end of this protocol to generate a new template.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Under Instrument Settings, start by selecting CytekAssaySetting. Lower gains for all channels by -80% by entering this into the All Channels % field. Please remember to do this for every laser line.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Vortex the vial of LEGENDplex™ Setup Beads 3: Raw Beads for seconds to resuspend the beads.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Transfer 400 μL of the Raw Beads to a new FACS tube.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Load the tube of Raw Beads onto the cytometer.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Acquire data with the instrument flow rate set to LOW. The LEGENDplex™ Experiment Template is set to have stopping criteria of 4,000 singlet events.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Adjust the FSC and SSC gains such that the 2 bead populations are on scale and fall within the gates provided in the LEGENDplex™ Experiment Template. You can find typical FSC and SSC gain settings here.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Verify that the 2 bead populations within the beads A and B gates are on scale on the R4 axis in the R4 vs. YG1/B4 plots found in the downloaded LEGENDplex™ Experiment Template.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
If your Cytek® instrument does not have a YG laser:
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
If your Cytek® instrument has a YG laser:
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Vortex the vial of LEGENDplex™ Setup Beads 2: PE Beads for 30 seconds to resuspend the beads.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Transfer 400 μL of the PE Beads to a new FACS tube.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Load the tube of PE Beads onto the cytometer.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Acquire data with the instrument flow rate set to LOW.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Verify that for Beads A, the bead population is on scale on the YG1 or B4 axis in the R4 vs. YG1/B4 plots found in the downloaded LEGENDplex™ Experiment Template.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Save settings as LEGENDplex™.
?. [Setting Up LEGENDplex™ for the First Time on Your Cytek® Instrument:]
Your Cytek® flow cytometer is now ready for sample acquisition.
?. Perform instrument start-up procedure and instrument QC per manufacturer’s recommendations.
?. Complete one of the following options:
?. Under Instrument Settings, select your saved LEGENDplex™ settings.
?. Your Cytek® flow cytometer is now ready for sample acquisition.
?. a. Option A Under the “Acquisition” module, click on “Template” and open the existing LEGENDplex™ Experiment Template.
?. b. Option B Under the “Acquisition” module, click on “My Experiments.” Find the previous LEGENDplex™ experiment that you ran, right click, and “Duplicate without Data”. This will create a new experiment with the same tube layout and acquisition criteria as your previous experiment. Open the newly created duplicated experiment.
?. [III) Instructions for Experiment Template Creation]
Under the Acquisition module, create a New experiment. A pop-up wizard will appear stepping you through how to create your experiment.
?. [III) Instructions for Experiment Template Creation]
First, enter the desired name and description (optional) for your experiment.
?. [III) Instructions for Experiment Template Creation]
You will need to add two or more fluorescent tags to continue. In this case, we will add PE and APC. Then click the Next button in the bottom right.
?. [III) Instructions for Experiment Template Creation]
You will now add the necessary Groups and Tubes. For the LEGENDplex™ assay, you will need to next create a Setup group, a Standards group, and a Samples group. See below for naming of your Setup and Standards beads. Then click Next to go to the next tab in the Create New Experiment wizard.
?. [III) Instructions for Experiment Template Creation]
You can skip the following tabs going over Markers and Keywords and skip straight to the Acquisition tab.
?. [III) Instructions for Experiment Template Creation]
Under the Analysis tab, you can select any pre-designed analysis worksheet, as well as stopping criteria. When starting to acquire your tubes, please ensure you collect no more than 5,000 events per tube.
?. [III) Instructions for Experiment Template Creation]
Once done with the Create New Experiment wizard, click “Save As” to create your experiment template.
?. [IV) Instructions for Setting Up Your Worksheet Template]
For your analysis worksheet, first gate around your bead singlets.
?. [IV) Instructions for Setting Up Your Worksheet Template]
Next, you will see a Beads A and Beads B population when gating off FSC-A and SSC-A.
?. [IV) Instructions for Setting Up Your Worksheet Template]
For both Beads A and Beads B, make sure you can see all events when looking at the R4 and B4/YG1 channels