After you compelted the experiement setup wizard, AutoGate takes to you the gating side of the analysis.

By default, the samples are organized based on the grouping done in the autocompensation setup wizard. You can change the naming of the samples or grouping by choosing Edit > Select Keywords. The grouping helps when you choose to apply gates for samples within the group or across groups.

It is important to understand the tree, as this will go a long way in completing your analysis with ease. Navigate through the menus and they are mostly straight foward.

gating Tree

The features are accessible via the right click menus, the toolbar and from the menus at the top of the screen.

gating tree menu bar

To view a sample, double click on the sample and it opens in a plot editor window.

If you are unsure or couldn't find what you are looking for, just ask GENIE, which is available on the lower left corner of the screen.

After completing the setup, if you wish to be view the (Auto) compensated numbers, select a sample and choose View > Compensation matrix.

View matrix

It brings up the compensation matrix.

comp matrix

If you realize there is a need to update the setup details, you can do so by clicking Edit > Redo compensation & setup

edit redo

Follow the prompt and it will take you back to the setup wizard

The samples in the gating tree are organized based on the grouping set in the experiment setup wizard. Grouping helps when you choose to apply gates for samples within in the group or across groups.

To change the grouping or the naming of the samples, click Edit > Select keywords

Ensure that you select Update tree now in the keywords window, to see the change reflected.

group samples

 

If the grouping that you need can't be achived using keywords, you can also manually create a grouping folder and move the samples to the folder.

Click File > New folder

new folder

Specify a name for the folder

new folder name

Drag the sample and drop it on the new folder icon

DND

It is helpful to declare the baseline sample within the group (to be expanded).

Right click on the baseline sample and choose Set as group's sample control (baseline)

declare baseline

AutoGate will respond by noticing if a sample in other group has a similar treatment and thus asks this question

declare other sample

This sets the base line in both time groups as the sample with a value of “no allergen” (in this example) for the keyword treatment.

AutoGate responds by highlighting both samples in bold to indicate that they are base line sample.

highlight baseline

AutoGate uses our statistically rigorous Density-Based Merging (DBM) to plot the subsets and their subsets in your samples. AutoGate's cluster analysis is based on this publication.

The plotting is automated. It is statistically correct to an unmatched degree. And you can use it to create gating models to apply to other experiments.

Our software gives you different ways to do what you want to do and a massive amount of useful information to help you do it. Give yourself time to take everything in.

To view the plots, double click on the baseline sample in the gating tree.

 AutoGate responds by

  • Opening the plot editor window and the path finder window.
  • Computing the bi-exponential’s W (width) parameter to set the logicle/linear region of the scale as accurately as possible. It does this by analyzing EVERY sample which uses the same compensation matrix. This only happens the first time that AutoGate opens a sample for each compensation matrix in an experiment.
  • Setting the X and Y axis to defaults automatically found during experiment setup
  • Clustering the data for all of the sample’s cells and the X and Y markers
  • Showing each cluster by a different color

Plot editor

 

Pathfinder helps to look aside/ahead into all stain parameters

plot editor click preferences

 

To change graph type or alter probability contour lines, click Preferences

plot editor view preferences

 

AutoGate gives you the tools to design the way your plot graphs look -- their colors, clusters, contours, gates, grids, and parameters. Design it all in no time with just a few clicks of your mouse.

Go to the upper right hand corner of the graph and click See Preferences. Your options will appear to the right side of the window.

As you can see, most of the settings are self explanatory. Below are some of the frequently sought after preferences.

 1. To view the labels for the clusters and or gates, click on the Lables drop down and select the required option

also, select the Arrow from all labels check box

cluster labels

2. You can also change the default graph type, as below.

graph type

 3. To change the level of cluster detail, click on the Cluster details icon on the toolbar, as indicated below

cluster details

 4. To set/change the % of outlier, choose Advanced preferences from the cluster details

Advanced preferences

 and choose % outlier from the Background if density drop down

Outlier setting

 5. You can also control the characteristics of Gating tree and Publication output by choosing from the Set preferences for... drop down, as highlighted below

preference target

Ensure the appropriate X and Y axis are set.

To set the axes you click on the down arrow button next to the X and the Y axis labels to activate a drop down of choices.

setx axis

 To change Y axis;

sety axis

Select the clusters either by;

  • Clicking on the cluster
  • Choosing from the cluster drop down from the tool bar

AutoGate responds by;

  • Drawing a black border around the green cluster to signify user’s selection.

select cluster

 

Select other clusters, as required.

Now, Click on Show preferences and choose Enlarge selected from the Gate label preferences drop down. You will notice that the total cell count (% of the samples) is shown now. Click Hide preferences

enlarge selected

 

To name the gate, click on the down arrow button.
name gate

 

Once you specify the name and click OK, AutoGate responds by restricting the plot window to ONLY the cells/events within your chosen cluster.

You will also notice that the created gate is reflected in the Path finder and Gating tree.

gate reflected

 

To create subgates, use the same actions for "Live dumped" gate above.

Subgate

 

Another sub gate added, as below

Subgate 2

Once you have the gates created for the baseline sample, it's now time to apply it across samples.

You can do this via;

1. Right click on the Plot editor window and choose Apply gates across samples

2. Click on the target button on the tool bar in the plot editor window

apply gate editor

 

3. Apply from the gating tree - via Right click or from the Edit menu

apply gate tree

 

Choose to Apply to all samples with compatible reagents

apply all samples

 

AutoGate then presents the below window for confirmation, along with the default EMD

Apply confirmation

The top entry field in the above window is the earth mover’s distance (EMD) threshold. By default AutoGate sets it to 0.08. The purpose of this is only visual - to tell AutoGate to highlight any sample that is greater than or equal to this.

threshold. AutoGate does not pre-determine this threshold. Instead, the researcher must arrive at an appropriate threshold by testing multiple experiments with AutoGate’s EMD measurement. The default of 0.08 was chosen after looking at several patients for the current basophil allergy of assay.

On clicking OK, AutoGate starts applying gates to other samples

 

applying gates

 

Once completed, it then prompts for confirmation on the output format

Show final statistics

 

On choosing show final gates of sequence first, AutoGate responds

  1. Quantifying the degree of difference/change between each sample’s final gate “Stimulated basophils” using the earth mover’s distance statistic (EMD)
  2. Showing the final results with this window. If a gate surpasses the EMD threshold AutoGate puts a check mark in column 1 and makes the font of the EMD column bold and italics.

show statistics

 

You can choose to copy the output, say, to Excel. Click on the Export button, choose Excel workbook and click Save

export statistics

 

Choose to embed images. The excel output will be similar to what is shown above.

Embed images

AutoGate will gate your clusters and subsets for you, and do it with far greater statistical rigor than you can. But, if at any time you want to DIY, AutoGate lets you gate by hand and mouse, much as you would in FlowJo.

Click the cluster you want to gate. To make it easier to see clearly, click on the zoom-in icon 

Go to the row of icons at the top of your plot window and click one of the geometic shapes – rectangular, polygon, or ellipse.

tools manual gate

Point your cursor on the cluster. A message will tell you which part(s) of the shape to click first. Put the shape in the subset you want to gate. Double-click. A window will appear, asking you to name the subset.

If you make a mistake at any point in the process, click the delete icon  and start over

The Path finder window;

  • Associates with a specific plot editor window
  • Shows the staining on all parameters for selected cells or for all cells in the associated plot editor window.
  • Depicts each parameter with a horizontal bar that uses pseudo color’s convention of showing where staining occurs most.
  • Uses a vertical red dashed line to indicate a user definable threshold for positive
  • Uses bold font for labels of parameters unused in the current gating sequence and normal font with a “>” prefix for labels of parameters used in the current gating sequence.

To access path finder anytime, click on the icon indicated in the below window.

path finder toolbar

 

It then opens up the Path finder window. 

pathfinder

 

Click on the tools icon to view the preferences

Path finder preferences

 

If you wish to go back to viewing the gating statistics, right click on the gating tree and choose Show gating statistics

menu statistics