TITLE: MPAPASS - Gating flow cytometry multiplex data
AUTHORS: Joshua Welsh, Sean Cook, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [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.
?. [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.
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]
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.
?. [Gating the Bead Populations]
Each of the distinct populations corresponds to a antibody coated bead that can be determined from the bead legend below:
?. [Gating the Bead Populations]
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]
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.
?. [Separating the Merged Population]
Creating the Derived Parameter:
?. [Separating the Merged Population]
In this new window, at the top, name the derived parameter 89,99 Sep.
?. [Separating the Merged Population]
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
?. [Separating the Merged Population]
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.
?. [Separating the Merged Population]
Gating the Merged Population:
?. [Separating 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.
?. [Separating the Merged Population]
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.
?. [Separating the Merged Population]
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]
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]
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.
?. [Exporting the Files]
Navigate to the file tab and click on the Export/Concatenate Populations button.
?. [Exporting the Files]
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.
?. [Exporting the Files]
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.csvRepeat if this occurs with the 89 bead population.