When you specify the experiment data files, AutoGate opens up the setup Wizard with sample and reagent details populated. You can review the details in the wizard and make changes, as required.

AutoGate provides two ways to navigate our experiment set-up wizard. Click the <Prev and Next> tabs at the bottom right of the windows. Or go to the To Do list at the left and click any of the enabled (darkened) titles. The blue arrows on the To Do list shows where you are. A white check mark in a green circle shows the windows you have completed and might want to revisit.

to do list

This is the first step of the setup.

In this window,

  • Confirm the target species.
  • Look over the other information.
  • Add any comments you want to save.
  • Choose to import from a prior AutoGate experiment,

Once done, click Next.

 

Describe experiment overview

This window displays the sample details. By default, only basic details are shown. To view samples count, particle type, marker or fluorphore information, click on >> More detais button on the top right corner

More details

In this window, you can verify/change;

  1. Each sample’s type: unstained, single-stained, multi-stained or standardization. To change the sample type, double click on the Type column and choose from the drop down
  2. stain type

  3. Click >>More details button on the top right window and double click on the Particle column to change. Click to choose either Beads or Cells. The first time you do this, the same choice will appear for all the relevant samples.
  4. samples particletype
  5. Each single stained sample’s
    • Associated channel
    • Fluorophore (optional)
    • Marker (optional)
    Again, double click on the relevant columns to change.

Click Next to move to Define stain sets and FMOs. Or go to the To Do list and click Create, manage, and assign keywords.

This step helps you to identify and group your samples as per your experiment needs.

Click to check Name samples or Group samples radio button. In either setting, the left-hand panel shows the column headers to display while the right-hand column lists those that are available. Double-click any item in either panel to move it across to the other panel. 

If the left-hand panel does not already show your currently selected column headers, move the ones you want from the right-hand panel. To change the order of column headers in the left-hand column, click any column header and drag it up or down.

Namedsamples

 

Click Group samples. Most of the commands work exactly as with identifying samples. Select the column headers you want to group together. 

Double-click the column headers you have selected. They will move into the left-hand panel.

Click to check Update table now. The grouping will automatically display in a new panel on the Characterize samples window and other tables.

groupsamples

 Click the X in the red circle to delete the grouping. Click the wrench icon for other available commands. 

For further options, click to check Show keyword controls.

Click Create new keyword to add a column header of your own. This small screen will display. 

Enter the name of your new keyword. Click OK. The name will display in the right-hand column of the keyword screen. New keywords will appear in magenta. Repeat until you have all the new keywords you want to create.

create keyword

To delete one or more user-defined keywords in either panel, click + CTRL to select them and Click Delete selected keyword(s). To edit a single user-defined keyword in either panel, click to select it and click Edit selected keyword. To enlarge the list of available column headers, click to check Show all keywords.

When you've finished identifying and group your keywords, click Done. Click Next on the Characterize samples window.

The define stain sets screen shows the number of stain sets added by default. To add more stain sets, click on the Select Number of stain sets drop down to choose the number of stain sets.

To review/complete the stain sets, click on Define button for the corresponding stain set.

click to define ss

AutoGate responds by showing the staining of each stain set on the channels of the instrument. Based on it’s scan of the experiment’s meta data in the sample files, AutoGate initially sets

  • The used checkbox for each channel it finds a reagent.
  • The marker & fluorophore values for each channel
  • The associated compensation controls: single-stained and unstained

If you wish to change the marker name, double click on the cell and choose from the marker/synonyms drop down.

edit marker

To specify dilutions for the reagent, click >>More details button at the top right corner which will display the additional details including Dilutions.

Double click on Dilutions to specify.

specify dilutions

Ensure that all details are correct/updated including dilutions and click on Save.

Once all stain sets are defined, you can hover the # of samples count to see the associated samples.

stainsets defined

If there is a FMO, it is automatically detected and defined, as in the example below

FMO stainset

In this step, you review and update the stain set associations with the samples.

Click to check or uncheck the boxes in the stain set and FMO columns to associate the samples where you want them.

Associate samples

When you've finished, click Next.

You are now all set to Run auto compensation. Click Run AutoComp in this screen.

run autocomp

Once successfully computed, it will prompt if you wish to see the compensation details or proceed to next step. 

To view the results, choose Inspect compensation details

View results

It pops up the results window, as below. Select the check boxes (Out of Range/Scatter Gatted/...) on the top window to see the relevant details in the graph.

comp results

You can also export the results to FlowJo by click on the Save matrices for Flowjo? checkbox at the bottom left window.

save for flowjo

The below screen lets you pre-define the X/Y axis display for the gating plots . Double click on the axis column and choose the reagent from the drop down

SetX Y

You are now set to run AutoGating. Click Start gating

On specying your sample data files, AutoGate opens up the setup Wizard with sample and reagent information populated from the data files.
You can choose to review the details or skip to start gating.

To start gating, click on the Start/Continue gating from the to do list, as below.

start continue gating

AutoGate experiment setup wizard can be largely customized.

To re-configure the table,

Right-click or Apple + click anywhere on the table to reconfigure it to suit your way of working. A small menu will display.

Say, To sort/arrange columns, right click to choose Manage columns > Sort/arrange columns

right click menu


To change values for all rows, double click on the column header and choose Change all rows

column header options

To avoid/skip the setup wizard for experiments that are the same type of staining, users could reuse the details from prior experiment by choosing "Choose previous AutoGate experiment" .

This will see the gating model automatically applied after the wizard exits

choose previous experiment