Welcome to the video tutorial for BioLegend’s cloud-based LEGENDplex data analysis software.

In this video, We will be showing you how to perform the basic steps needed to analyze a LEGENDplex data set

Please note: Users are strongly encouraged to read the entire software manual for a complete explanation of all program features

For the purposes of this tutorial, we will begin on the software home page, which is displayed upon logging in to the program.

To begin let’s create a new experiment

Click the purple folder icon to the left of your screen

Next, click the plus icon

Enter your experiment name and click the create button

Find your particular LEGENDplex assay from the bead panels pull down menu

This will then populate all the bead ID fields associated with the assay

For mix and match assays, please click the trash can icon to remove any unnecessary targets

If you are analyzing a custom LEGENDplex assay, select custom panel from the drop down menu.

Note: You will need to manual define the bead IDs for each custom panel

Next, enter the top standard concentrations into the MAX column. These values can be found on the CoA included with your LEGENDplex assay.

Click save to proceed to data upload steps

If required, adjust the parameters at the top of the screen to match your assay.

Note: The default settings match those described in the LEGENDplex assay manual and typically do not need to be adjusted.

Click the ‘select standards FCS files’ button

Navigate to the appropriate folder on your computer, and then select all standard curve files from your assay and click open

Ensure that the files are ordered in increasing concentration going down the file list with C0 at the top and C7 at the bottom

Adjust the file order if necessary by clicking and dragging the files into the appropriate order

Select both the PE reporter channel and APC bead channel from the pull down menus to match your flow cytometer settings

Click the purple upload button to upload your standard curve files to the cloud server

Next, we will upload any experimental samples

Click the select samples fcs files button

Navigate to the appropriate folder on your computer, and then select all sample files from your assay and click open

Should you wish to define samples as replicates of one another please complete the following steps:

Adjust the parameters in the replicate controls box to match your assay files and then click apply

This will apply the replicate control settings to your sample files

Note: The sample name field is entirely editable should you wish rename your samples beyond the default provided by the software

All files with the same sample field name will be treated as replicates by the software

Next, please input any sample dilution factors

You can manually enter values for each file in the dilution column

Alternatively you can apply a global dilution factor to all samples by entering a value into the dilution box

Click the upload button which is found below the sample files list

Wait for the program to gate all files then click review to proceed to the gating review step

Select all file you wish to review from the list displayed by the software

Note the default setting includes all uploaded files

Click review

For the file displayed, ensure gating accuracy in the sizing plot as well as both the A and B bead univariate APC plots

In the unlikely event of a gating error please do the following

Click any plot to enable manual adjustments and fix the gating error

Remember to click save to apply any changes

Once you are satisfied with the gates, check the reviewed box to the upper left of the screen

The cart icon above the box will track the reviewed status of all uploaded files

Use the right and left cursors found on the side of the plot images to scroll through and review all files, making any necessary adjustments

Once you are finished, Click the results tab to move to the quantitation results

At the top of the screen, you will find a graphical display of the curves fitted by the software’s 5 parameter logistic curve fitting algorithm to your LEGENDplex assay’s standard curve files

You can toggle between different curves by clicking the desired analyte’s name at the top of the results table

Mouse over items on the graphs to see further details and definitions

The quantitation results are listed by analyte columns in the results table

You can toggle to the bead count, PE-MFI, and replicate %CV tables by clicking their respective circles

When finished, Click the report button to download your LEGENDplex assay data into an excel report

One of the most exciting features in the software is the ability to instantly share your experiment workspace with other user accounts and BioLegend technical service

To access this functionality, click the Click the purple folder icon

Find you assay and then click the 3 bar icon to the right

Click share, then click the text field

If you would like to share your data with BioLegend technical service, simply check the box now visible

If you would like to share your data with another user, type their account email address in the field and hit enter

The software will automatically send an email to the user informing them that you have shared your data

You may un-share data at any time by repeating this procedure and clicking the ‘x’ next to an account you have shared data with

Finally, should you ever wish to return to the landing page, click the BioLegend logo at the top left of the screen.

Please remember to read the user manual for a complete explanation of all the features in the cloud-based LEGENDplex data analysis software

Do not hesitate to contact us if you have any questions, and best of luck with your experiments.