Live Dead Cell Analysis Protocols

Introduction

The live dead cell analysis protocols consist of four protocols that allow you to analyze various combinations of cellular markers for total, live and dead cells.   All protocols report the total cell number, the number of live cells and the number of dead cells.

Each protocol follows the same workflow, the number of steps is dependent on the cellular markers used in the experiment.

Live Dead Cell Protocol Information

  • Live Dead Analysis: Total, Live, and Dead

The protocol determines cell viability by measuring the populations of total, live, and dead cells.  The Total, Live and Dead protocol requires a multi-channel fluorescent image containing 1) a stain to determine the total cell population, 2) a stain to determine the number of live cells, and 3) a stain to determine the number of dead cells.

Any cell that contains both live and dead cell stains scored as dead.

  • Live Dead Analysis: Total and Dead

The protocol determines cell viability by measuring the populations of total and dead cells.  The Total and Dead protocol requires a multi-channel fluorescent image containing 1) a stain to determine the total cell population, and 2) a stain to determine the number of dead cells.  The # of Live cells = # Total Cells - # Dead Cells.

  • Live Dead Analysis: Total and Live

The protocol measures cell viability by measuring the population of total and live cells.  The Total and Live protocol requires a multi-channel fluorescent image containing 1) a stain to determine the total cell population, and 2) a stain to determine the number of live cells.  The # of Dead cells = # Total Cells - # Live Cells.

  • Live Dead Analysis: Live and Dead

The protocol determines cell viability by means of measuring the population of live and dead cells.  The Live and Dead protocol requires a multi-channel fluorescent image containing 1) a stain to determine the number of live cells, and 2) a stain to determine the number of dead cells. The Total number of cells = # Live Cells + # Dead Cells.

Live Dead CellTotal, Live, and Dead Setup: Step 1

The live dead cell Total Live and Dead protocol will be used as an example workflow for all live dead cell protocols.

For additional information on setting up protocols, please refer to the Protocols Help section: Protocol Setup.

  1. Load your image dataset. An example of a dataset suitable for wound healing analysis can be found in the File/Open/Demo Images in the Protocol Live Dead folder.  The following instructions are based on using the demonstration image.

  2. Select the Count/Size tab. 

  3. Press the Protocols button to display the protocol browser.

  4. Load the default Live Dead Analysis: Total, Live, and Dead protocol located in the Cell Biology collection.

  5. The following steps are based on using the using a pre-configured protocol for the demo dataset.  You may also use your own images and settings as needed.

  6. Select the Reset drop down and press the Load Demo button.  The Open Demo Image option should also be checked to open at the same time.

  7. Press the Sync button to load the active image channels.  Select the appropriate channels in the “Total”, “Live” and “Dead” cell combo-boxes.

  8. Select the Interactive Mode check box and press the Next Step button.

Live Dead Cell Total, Live and Dead: Segmentation: Step 2

For more information on segmentation and recipe creation, please refer to the Protocols Help section: Protocol Segmentation.

  1. Segmentation

    1. Adjust the total cell marker segmentation using the histogram slider or pressing the Auto-bright segmentation button to automatically segment the cells. 

      When using Automatic Bright thresholding, the auto-thresholding may be adjusted using the Standard Deviation offset (which is located to the right of the Auto-bright segmentation button).  The Standard Deviation offset will help correct for well-to-well intensity variation. Press the Set Thresholds OK button in the Interaction popup when done adjusting the segmentation

  2. If Threshold Segmentation is not effective with your data, the segmentation method may be changed to Smart Segmentation.

  3. Adjust the segmentation options as needed to identify the cell marker.  Press the Apply button to save the changes to the protocol.

    1. Grow: sets the object outline growth size in pixel.   

      Note: Some protocols contain predefined settings and may need to be adjusted to suit your data

    2. Min Area: Sets the minimum object size to be included in the analysis.  The Edit Range button is used to set the minimum and maximum object size using and interactive histogram to set the limits.

    3. Fill Holes: when selected, any holes within your objects will be filled.

    4. Split: select either Boundary Shape or the Watershed cell splitting methods to separate joined cells or None.

    5. Clean Borders: removes cells touching the edges of images if selected.

  4. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Live Dead Cell Total, Live and DeadSegmentation: Steps 3 and 4

In Steps 3 and 4, the protocol respectively segments the live cell marker followed by the dead cell marker.  The total cells identified in Step 1 are outlined in each step for clarity.   

  1. Once the fluorescent signal has been segmented, press the Set Thresholds OK button.

    Additional segmentation options for removal of objects by size may be applied to remove non-specific fluorescence.   Press the Segmentation Options Apply button if changes have been made. 

  2. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Live Dead Cell TLD Total Protocol: Results

For more information on configuring and showing analysis results, saving protocols, or running the protocol on selected wells, please refer to the Protocols Help section: Configuring Results.