Endpoint apoptosis using Caspase 3/7 with Hoechst

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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, Bright field and Blue channels
Hypothesis By measuring the number of Caspase 3/7 positive cells, we can determine the percent of apoptotic cells in the population

Celigo Setup

Plate Type Greiner 655090 96-well black wall clear bottom
Scan Channels Green, Brightfield and Blue
Resolution 1 µm/pixel
Scan Area Whole well
Analysis Method Target 1 + 2 + Mask
Scan Frequency End Point
Scan Time ~15 minutes

Assay Protocol and Plate Setup

Goal:

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

Protocol:

  • Seed MDA-MB-231 at 10,000 cells/well and Jurkat cells at 20,000 cells/well and allow to incubate overnight
  • Add Staurosporine at 3 µM final concentration per well and allow to incubate for 4-6 hours
  • After incubation is completed, prepare in PBS a 2X concentration of Caspase 3/7 and Hoechst
    • Nexcelom, Cat# CSK-V0003-1
  • Remove 100 µL of media from all plate wells.
  • Add 100 µL of 2X concentration of Caspase 3/7 and Hoechst and incubate for 30 mins at 37° C
  • Image the plate using the Celigo image cytometer

endpoint apoptosis plate set up

Results

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

  • Total number of nucleated cells was determined by counterstaining the cells with Hoechst
  • Total number of apoptotic cells was determined by counting the nucleated cells stained with green Caspase 3/7 reagent
  • Determined the percent of apoptotic-positive cells

Plate-Level Data View allows a quick observation of the total number of cells and green Caspase 3/7 positive cells, as well as percent Caspase 3/7 positive cells. Currently displaying percent Caspase 3/7.

endpoint apoptosis plate level data

Whole-well view allows high-resolution observation of images and at zoomed levels.

endpoint apoptosis whole well view

Gate Plots for Caspase 3/7 Positive Cells:
Apoptosis Caspase 3/7 gating analysis of an MDA-MB-231 and Jurkat cells

MDA-MB-231 Adherent:

endpoint apoptosis MDA MB 231 adherent

Jurkat Suspension:

endpoint apoptosis jurkat suspension

Graphs

  • Cell counts for adherent, MDA-MBA-231 and suspension, Jurkat cells plotted
  • Using Hoechst cell counts as a total, percent Caspase 3/7 positive cells are plotted on bar graphs

endpoint apoptosis 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, Caspase 3/7, and Hoechst fluorescent images of an entire 96 well plate took ~ 15 minutes
  • Performing an endpoint apoptosis assay using Caspase 3/7 with Hoechst allows for the enumeration of the total number of nucleated cells and the total number of Caspase 3/7 positive cells, as well as to determine the percent of apoptosis.
2018-10-11T19:12:15+00:00