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SubtitleText Viewing t-SNE plot of Astrocytoma 1 AnchorName Viewing single-sample t-SNE
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The t-SNE plot is in 3D by default. You can rotate the 3D plot by lef-clicking and dragging your mouse. You can zoom in and out using your mouse wheel. The 2D t-SNE is also calculated and you can switch between the 2D and 3D plots using the Plot style radio buttons. 

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SubtitleText Viewing t-SNE plot of Astrocytoma 2 AnchorName Viewing single-sample t-SNE (2)
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The goal of this experiment is to compare malignant cells from two different glioma subtypes, astrocytoma and oligodendroglioma. To do this, we need to identify which cells are the malignant cells we want to include and which cell are the normal cells we want to exclude. 

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SubtitleText Selecting color by gene expression AnchorName Selecting color by gene expression
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The cells will turn black and a text box Gene ID will open below the drop-down box. 

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SubtitleText Coloring cells by CD14 expression AnchorName Selecting gene
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The cells will be colored from black to green based on their expression level of CD14, with cells expressing higher levels more green (Figure 8). CD14 is a known marker for microglia and macrophage cells, used by the authors of the original study to classify microglia/macrophage cells. 

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SubtitleText Cells colored by CD14 expression AnchorName Colored by CD14
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In Partek Flow, we can color cells with up to three genes at a time. We will now add a second gene, MOBP. 

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SubtitleText Coloring cells by MOBP and CD14 AnchorName Coloring by two genes
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Relative expression of the two genes for selected cells can be visualized on the legend. 

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SubtitleText Selecting a group of cells using the 3D lasso tool AnchorName Selecting a group of cells
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Selected cells are shown in bold and unselected cells are dimmed.

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SubtitleText Viewing expression levels for a group of cells AnchorName Viewing expression levels for a group of cells
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Numerical expression levels for each gene can be viewed for individual cells. 

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SubtitleText Viewing expression levels for an individual cell. The dots on the legend indicate the expression level of the selected cell. AnchorName Viewing expression levels for a cell
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Classifying cells from individual samples on t-SNE plot

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SubtitleText Selecting malignant cells AnchorName Selecting malignant cells
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• Select Classify selection (Figure 14)
  
  

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SubtitleText Classifying selected cells AnchorName Classifying cells
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A dialog to give the classification a name will appear. 

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SubtitleText Classifying selection AnchorName Classifying cells
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Once cells have been classified, the classification is added to the Classifications section of the panel. The number of cells belonging to the classiciation is listed; in Astrocytoma 2, there are 253 malignant cells (Figure 15). 

 

 

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SubtitleText The number of cells in each classification is displayed in the classification section. AnchorName Viewing classifications
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Classifications made on the t-SNE plot are retained as a draft until you exit the t-SNE task report. The Save classifications button runs a task, Classify cells, which generates a new Classified cells data node. In this tutorial, we will classify malignant cells for each sample before we save the classifications, but if necissary, you can run Classify cells for one sample, exit the t-SNE task report, and continue classifying the next sample later starting with the Classified cells data node. 

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SubtitleText Classifying malignant cells AnchorName Classifying cells
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• Select Classify selection 
• Type Malignant or select Malignant from the prompt (Figure 17)
• Select Save
  
  

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SubtitleText Adding cells in a second sample to an existing classification AnchorName Classifying cells as an existing classification
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• Repeat these steps for each of the 5 astrocytoma and 3 oligodendroglioma samples

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SubtitleText Navigating to the classification summary AnchorName Navigating to classification summary
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The classifications summary lists every sample, the number of cells in the sample, and the number of cells in each classification (Figure 19).

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