LEGENDplex™ Cloud-Based Software Tutorial
Description: Learn how to analyze your LEGENDplex™ multiplexing data with BioLegend’s free cloud-based software. We walk you through the process step-by-step so you can generate meaningful results from your experiments.
Transcription:
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.
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.
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