Standard Operating Procedure (SOP) for Cell Counting using ImageJ

Cell counting is a fundamental technique in cell biology, crucial for understanding cellular processes such as proliferation, differentiation, and apoptosis. In the context of cancer research, proliferation assays are essential since cancer cells often exhibit uncontrolled growth and division. Counting cells can provide valuable insights into the efficacy of cancer treatments aimed at reducing cell numbers or inhibiting their growth. This Standard Operating Procedure (SOP) outlines a method for counting cells using ImageJ, a popular open-source image processing program. By quantifying cell numbers, researchers can assess the impact of various treatments on cell proliferation, contributing to the development of more effective cancer therapies

Open ImageJ and Load the Image

• Launch ImageJ.
• Navigate to File -> Open.
• Select and open the image file containing the cells to be counted.
  
  

Convert the Image to 8-bit

• Go to Image -> Type -> 8-bit to transform the image into an 8-bit grayscale image. This step is crucial for enhancing contrast and facilitating the identification of individual cells.
  
  

Create a Region of Interest (ROI)

• Use the circle selection tool to create a circular ROI and select a portion of the well.
• Navigate to Analyze -> Tools -> ROI Manager.
• Right-click on the ROI within the ROI Manager and choose Save to store the ROI for future analysis.

Duplicate the Image

• Right-click on the image and select Duplicate to create a copy of the original image. This allows for processing without altering the original data.
  
  

Set a Threshold

• Go to Image -> Adjust -> Threshold (or use the shortcut Ctrl + Shift + T) to apply a threshold. Adjusting the threshold helps to distinguish cells from the background.
  
  

Apply the Watershed Algorithm

• Navigate to Process -> Binary -> Watershed. The watershed algorithm helps to separate cells that are touching, making individual cells more distinguishable.
  
  

Analyze Particles

• Select Analyze -> Analyze Particles. This function quantifies and categorizes particles (cells) based on size and shape, effectively counting them.
  
  

Check the Results

• In the results window, examine the last row, first column to find the number of cells (#cells).
  
  

Measure Area of the Well and ROI

• To measure the area of the entire well and the selected ROI, go to Analyze -> Measure (or use the shortcut Ctrl+M).
  
  

Calculate Cell Density

• To calculate the cell density within the selected area, use the formula:

• This calculation adjusts the cell count to account for the difference in area between the entire well and the selected ROI, providing a normalized measure of cell density.

By following this SOP, researchers can accurately count and assess the density of cells in a given sample. This process is invaluable for experiments aimed at understanding cell proliferation, especially in the study of cancerous tissues where cell growth is a primary concern.