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The t-SNE plot will open to the first sample in the data set, Astrocytoma 1 (Figure 4). Please note that the appearance of the t-SNE plot will differ each time it is drawn so your t-SNE plots will look different than those shown in this tutorial; however, the cell-to-cell relationships indicated will be the same. 

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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The cells will turn black and a text box Gene ID will open below the drop-down box. 

• Type CD14in Type CD14 in the Gene ID text box
• Select CD14 from the list of genes in the data set (Figure 7)
  
  

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The cells will be colored from black to green based on their expression level of CD14, with cells expression expressing higher levels being 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. 

In Partek Flow, we can color cells with up to three genes at a time. We will now add a second gene, MOBP. 

• Select the Image Added icon next to CD14
• Type MOBP in the new Gene ID box
• Select MOBP from the list of genes in the data set

Cells expressing MOBP are now colored red and cells expressing CD14 are colored green. Cells expressing both genes are colored yellow, while cells expressing neither are colored black (Figure 9).

Relative expression of the two genes for selected cells can be visualized on the legend. 

• Activate the 3D lasso tool by selecting Image Added 
• Draw a circle around the cluster of red cells (Figure 10)
  
  

Selected cells are shown in bold and unselected cells are dimmed.

The relative expression of the two genes for the selected cells will be shown on the legend as dots (Figure 11). 

Numerical expression levels for each gene can be viewed for individual cells. 

• Switch modes by selecting Image Added
• Select a cell by pointing and clicking

The expression level for that cell is displayed on the legend for each gene (Figure 12). 

• Deselect the cell by clicking on any black space on the plot

Expression values can also be viewing by selecting Gene Expression from the Label by drop-down menu and mousing over a cell. 

Classifying cells from individual samples on t-SNE plot

Now that cells are colored by expression of a microglia/macrophage marker, CD14, and a oligodendrocyte marker, MOBP, we can classify any cell that does not fall into one of these two groups as malignant cells. Because t-SNE groups cells that are similar across the high-dimensional gene expression data, we will consider cells that form a group with CD14 or MOBP-expressing cells as same cell type, even if they do not express the marker gene.

Starting with the Astrocytoma 2 sample, we can classify the malignant cells in each sample. 

• Activate the 3D lasso tool by selecting Image Added
• Draw the lasso around the cluster of black cells and click the circle to close the lasso (Figure 13). You may need to switch to selection mode and rotate the 3D plot to select only cells from the black cluster
  
  

• Select Classify selection (Figure 14)
  
  

• Name the classification Malignant 
• Select Save (Figure 15)
  
  

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). 

• Select Next to move to the next sample, Astrocytoma 3
• Rotate the 3D t-SNE plot to allow you to select only cells from the black cluster
• Activate the 3D lasso tool by selecting Image Added
• Draw the lasso around the cluster of black cells and click the circle to close the lasso (Figure 16). 
  
  

• Select Classify selection 
• Type Malignant or select Malignant from the prompt (Figure 17)
• Select Save
  
  

• Repeat these steps for each of the 5 astrocytoma and 3 oligodendroglioma samples

Once all samples have been classified, it is useful to check the number of cells in each sample assigned to each classification. 

• Select Classification summary (Figure 18)
  
  

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

With the malignant cells in every sample classified, it is time to save the classifications.

• Select Save classifications 
• Select Save when asked to confirm

The pipeline view will open and the Classify cells tasks will run, generating a Classified groups data node (Figure 20).