Kinetic apoptosis using Caspase 3/7

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Purpose Perform apoptosis assay on MDA-MB-231 and Jurkat cells
Existing Method(s) Flow cytometry
Target Cell Type MDA-MB-231 and Jurkat cells
Experiment Plan Scan plate using Green and Bright field channels
Hypothesis By measuring the number of Caspase 3/7 positive cells, we can determine the counts of apoptotic cells in the population

Celigo Setup

Plate Type 96-well Greiner 655090 black wall clear bottom
Scan Channels Green and Bright field
Resolution 1 µm/pixel
Scan Area Whole well
Analysis Method Target 1 + 2
Scan Frequency 0h, 2h, 4h, 6h and 8 hours
Scan Time ~15 minutes

Assay Protocol and Plate Setup

Goal:

Detect and quantify apoptotic cells using Caspase 3/7 staining in adherent MDA-MB-231 and suspension Jurkat cell lines

Protocol:

  • Seeded MDA-MB-231 at 10,000 cells/well and allowed to incubate overnight
  • Seeded Jurkat cells at 20,000 cells/well on the day of the experiment
  • Added Staurosporine at 3 µM final concentration and Caspase 3/7 substrate at 4 µM final concentration per well and allowed to incubate for 8 hours at 37° C
  • Imaged the plate every two hours using the Celigo image cytometer for a total of 8 hours

Plate Setup

kinetic apoptosis plate set up

Results

Drug-treated MDA-MB-231 and Jurkat cells showed an increase in Caspase 3/7 positive cells

  • Bright field images were captured to monitor cell health and morphology
  • The total number of apoptotic cells was determined by counting the cells stained with green Caspase 3/7 reagent

Plate-Level View allows for quick observations of the total number of green Caspase 3/7 positive cells. Shown below are typical results of apoptotic (Caspase 3/7 positive) cells after 8 hours of drug treatment.

kinetic apoptosis plate level data

Whole-well view allows for observation of high-resolution images.

kinetic apoptosis whole well images

kinetic apoptosis whole well images-2

Graphs

  1. In Microsoft Excel, create averages and standard deviations of the control and drug-treated wells.
  2. Generate a “Bar graph” comparing 3 µM Staurosporine to the control over 8-hour time course. In this example, the average of 12 data points were plotted.

kinetic apoptosis bar graphs

Conclusion

  • The Celigo successfully performed Caspase 3/7 apoptosis assay using MDA-MB-231 and Jurkat cell lines
  • Acquisition of high-resolution bright field and green Caspase 3/7 fluorescent images of an entire 96 well plate took ~ 15 minutes
  • Performing kinetic apoptosis assay using Caspase 3/7 allows for the enumeration of Caspase 3/7 positive cells over the time
2018-10-11T18:58:01+00:00