Sep 19, 2020
Version 1
MPAPASS - Gating flow cytometry multiplex data V.1
- 1Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health
- Translational Nanobiology Section
Protocol Citation: Joshua A Welsh, Sean M Cook, Jennifer Jones 2020. MPAPASS - Gating flow cytometry multiplex data. protocols.io https://dx.doi.org/10.17504/protocols.io.bjnwkmfe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2020
Last Modified: June 29, 2023
Protocol Integer ID: 40374
Disclaimer
This protocol summarizes key steps for a specific type of method, which is one of a collection of methods and assays used for EV analysis in the NCI Translational Nanobiology Section at the time of submission of this protocol. Appropriate use of this protocol requires careful, cohesive integration with other methods for EV production, isolation, and characterization.
Abstract
This is collection contains the protocols required for each step in the mpapass software pipeline for performing stitched multiplex analysis. This is one of a number of protocols in the pipeline for using the mpapass software package and is applicable to the latest release of the software.
Materials
FlowJo
Importing the Files into FlowJo
Importing the Files into FlowJo
Import the desired files into the FlowJo workspace using either the Add Samples (red box) button under the FlowJo tab (blue box), or simply drag the desired files into FlowJo.
Note
It is recommended that the fluorescence parameters be calibrated into standard units of 'molecules of equivalent soluble fluorophore' (MESF) to aid in reproducibility of data. This can be done using FCMPASS software in combination with commercially available MESF beads. See the FCMPASS software for further information at https://nano.ccr.cancer.gov/fcmpass
Gating the Bead Populations
Gating the Bead Populations
Double-click on any of the samples in order to bring up a scatter plot. Change the parameters of the scatter plot to FSC-A and SSC-A channels and gate around the single events using a rectangular gate.
In this example, the A1.fcs sample was used to gate for singlets.
Double-click on the newly gated Singlets population to open a new scatter plot. Change the parameters to the FITC-A and PE-A channels, and a pattern similar to the figure below should be seen.
There are supposed to be 39 distinct bead populations, however only 38 can be seen due to the merging of the 89 and 99 bead populations.
Each of the distinct populations corresponds to a antibody coated bead that can be determined from the bead legend below:
Gate each of the bead populations using a elliptical gate and label the populations according to the bead legend in step 4. For the merged population, gate around the entire population--later the population wil be separated.
Separating the Merged Population
Separating the Merged Population
On select cytometers it has been found that some lots of Miltenyi exosome multilpex beads do not adequately separate populations 89 and 99. If this is not the case proceed to the next section.
The FlowJo workspace should now have subpopulations under the Singlet population for each bead population as well as the merged population.
Click on the sample used to gate the bead populations (the highlighted A1.fcs sample in the figure below) and then navigate to the tools tab (blue box).
Click on Derive Parameters (red box) which will bring up a new window.
Creating the Derived Parameter:
In this new window, at the top, name the derived parameter 89,99 Sep.
Using the Insert Reference button (red box), choose the V-525-A channel. A string of characters will appear in the text box above.
Click on the division button next to the Insert Reference button.
Once again using the Insert Reference button (red box), now choose the V-660-A channel.
A plot similar to the figure above should be now be seen
Click on the collapse/open arrows for the Scale tab (denoted by a red arrow).
Choose the Linear scale and set the Min to 0 and the Max to 10.
Now click OK, and the derived parameter should appear beneath the chosen sample.
Gating the Merged Population:
Click on the 89_99 subpopulation to open a new scatter plot.
Choose 89,99 Sep derived parameter from the dropdown menu on the X-axis and the histogram option on the Y-axis.
A plot similar to the figure below should be seen.
Click on the Bisector Tool (red arrow) to gate the two bead populations. Make sure to choose a point where the two populations are distinctly separated.
The populations should be relabeled by right clicking the populations in the FlowJo workspace. The upper population should be labeled 99 while the lower population should be labeled 89.
Applying the Gates to Each Sample
Applying the Gates to Each Sample
In the FlowJo workspace, highlight all the gated populations and the derived parameter.
Now drag them into All Samples Group above (red arrow).
All files in the FlowJo workspace should now be gated exactly how we gated our chosen sample. This will be reflected in the FlowJo workspace as all samples should now have the same populations.
Exporting the Files
Exporting the Files
Now that all our samples have been gated, it is time to export the data from FlowJo into .csv files.
For any of the samples select all the bead populations to export, then under the Edit tab (blue box), click the Select Equivalent Nodes button to select all bead populations from every sample to export.
Navigate to the file tab and click on the Export/Concatenate Populations button.
Change the format to CSV - Scale Values.
Uncheck 'Include Header'
Change the Destination to desired folder.
Click on Custom Set of Parameters and then the View/Edit button directly next to it. Choose the APC parameter from the pop-up list.
Finally click Export to generate the .csv files in the destination folder.
In preparation for the MPAPASS software, you will have to manually rename the 99 bead population from the export suffix of 89_99_99.csv to simply _99.csv
Repeat if this occurs with the 89 bead population.