Assay Show: Use Celigo to perform a kinetic apoptosis assay using an adherent breast cancer cell line
Transcript Assay Show 3: Kinetic apoptosis assay that using an adherent breast cancer cell line.
Welcome to the assay show. This video will show you how to perform a kinetic apoptosis assay on the Celigo image cytometer using caspase 3/7 and Hoechst reagents
Let me first take a moment and describe the assay principle.
- DEVD is a caspase 3/7-specific sequence that is coupled with a DNA dye molecule.
- This substrate can freely defuse across the cell membrane in live cells.
- Once inside apoptotic cells, the caspase 3/7 protein recognizes and cleaves the DEVD sequence and releases the DNA probe.
- Once the probe enters the nucleus it binds to the DNA producing a bright green fluorescent signal.
After staining, the Celigo was used to acquire whole well bright field and Caspase 3/7-Green images.
The Celigo software automatically analyzes the captured images and reports the total number of green, caspase positive cells in the entire well. The captured bright field images were not analyzed but were used to monitor cell morphology.
Today I will show you a kinetic apoptosis assay that was performed using an adherent breast cancer cell line MDA-MB-231 that was treated with 3 micromolar staurosporine.
To achieve best accuracy of your cell plating, first measure the cell concentration by using a Cellometer automated cell counter.
Mix 20 microliters of cell sample and 20 microliters of trypan blue.
Load 20 microliters of stained sample into the Cellometer chamber slide and perform a cell count to acquire cell number, concentration and viability of your sample. Based on the measured concentration of your cells, adjust the volume and plate 10,000 cells per well in a volume of 200 microliters per well and allow the cells to incubate overnight.
On the next day, remove all media from the wells and add 100 microliters of 2-times concentrated 3 micromolar staurosporine drug treatment and 100 microliters of 2-times concentrated Caspase 3/7 staining solution to each well. Allow the plate to incubate at 37 degrees Celsius for 30 minutes and image the plate to acquire time point zero. Subsequently image the plate at 2 hrs, 6 hrs and 8 hrs after drug treatment.
The entire plate with whole-well imaging is captured in 15 minutes.
The analyzed results are displayed in a plate-based format showing a thumbnail picture and percent of apoptotic cells for each analyzed well.
Let’s take a closer look at a treated sample in well D3. By double clicking on a well the whole-well image appears for review.
We can zoom in to look at the cell morphology in the bright field image and examine the staining of caspase positive cells. By using the drop-down menu, we can quickly look at and compare samples captured at different time points
Direct cell counting of Caspase positive cells allows for the real time monitoring of apoptotic events on a per-well basis
All the data can be exported to excel as a .CSV file in a plate-based layout. Each file that is exported to excel contains the number of caspase 3/7 positive cells. Generated bar graphs show a time dependent increase in the number of caspase 3/7 positive cells in the staurosporine treated samples.
These and other assays are routinely performed on the Celigo To learn more or schedule a free in-lab demonstration call us or visit nexcelom.com