Obtaining intensity measurements from double-positive cells
#1
A user asked us this question recently, and I thought that my answers might be of interest to other users.

This is for a 3-color assay, with nuclear stain (DAPI) and 2 stains of interest (green/FITC and red/TR).
User wants counts/percentages of the single-positive (FITC-positive, TR-positive) and double-positive cells.
User also wants sum or mean of the intensity output only from the double-positive cells.

The Multi Wavelength Cell Scoring (MWCS) module will give the desired counts and percentages, but the intensity outputs are only available for all cells, or cells positive or negative for individual channels (e.g. intensity for all FITC-positive cells).  This module does not provide a summary intensity measurement from double-positive cells.

In this thread I will show you two methods to generate this output.
1. Use the MWCS module and using Export Measurements to filter the cellular data in order to calculate intensity only from cells that are double-positive.
2. Use a custom module.
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#2
Multi Wavelength Cell Scoring (MWCS) analysis:

The normal output from MWCS can include intensity of individual channels for all cells, or cells positive or negative for one channel, but not intensity measurements restricted to double positive cells.  

You can calculate this by saving the cell-by-cell data from MWCS analysis, and then configuring your Export Measurements appropriately.

1. Go to Screening menu > Plate Data Utilities > Export Measurements

2. Select option to export Cell Measurements.
   

3. Select measurement set of interest.
   

4. Add a filter to only select double-positive cells.
   

5. Select the intensity measurements of interest and how to calculate them (sum or mean over well or site).
   

6. Define the saving options to export the data as a tab-delimited .txt file.

7. Open the .txt file in Excel to review.
   

8.  Perform a separate Export Measurements step to output the counts and percentages of the FITC positive, TR positive, and double-positive cells as desired.

For more information on the Multi Wavelength Cell Scoring module and its outputs, or on using the Export Measurements function, please see our user guides and reference materials on our Knowledge Base.  You will need to be signed into your KB account.  Please contact Molecular Devices tech support if your account cannot access these materials.
Imaging User Guides
Imaging Reference Materials for MX5/MX6
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#3
Create a custom module:

1. Setup – make sure your DAPI, FITC, and TR images are available
2. Cell Scoring with DAPI and FITC – name the output masks appropriately.
   

3. Cell Scoring with DAPI and TR – use the identical nuclear/DAPI settings as in step 2.
   

4. Logical Operations OR to combine the FITC Nuclei and Cytoplasm to give you FITC Cells.
   

5. Logical Operations OR to combine the TR Nuclei and Cytoplasm to give you TR Cells.
   

6. Keep Marked Objects with positive FITC cells and positive TR cells to find double positive cells.
   

7. For an “All Cells” count, we need to make an “All Cells” mask.  This will take two steps.  First, Logical Operations OR on Positive FITC Cells and Positive TR Cells.
   

8. Next, Logical Operations OR on FITC or TR positive Cells and one of the negative nuclei to give All Cells.
   

9. Finally in the Segment steps, to restrict the output to summary data to get counts/averages/sums, we create a Simple Threshold mask over the whole image (any of the images).  This is optional.
   

10. In the Measure tab, Objects to measure will be the Whole Image Mask, with no measurements enabled.
Any image can be selected here.  (Alternatively, if you want cell-by-cell data, you can use the “All Cells” mask here.  The counts will appear differently if you do this.)
   

11. Add a feature group and select the All Cells mask.  The only measurement selected is Features Count.  (If you are using “All Cells” mask as the objects to measure, omit this step).
   

12. Add a feature group and select the Positive FITC Cells mask.  The only measurement selected is Features Count.
   

13. Add a feature group and select the Positive TR Cells mask.  The only measurement selected is Features Count. 
   

14. Add a feature group and select the Double Positive Cells mask.  Select the FITC image and select Features Count, plus the FITC intensity measurements that you want.  Also under this mask select the TR image and select the TR intensity measurements that you want.
   
   

15. Name, save and test run your custom module.
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