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Kinetic viability using DRAQ7

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Purpose Perform kinetic viability assay on MDA-MB-231 and K562 cells
Existing Method(s) Cell Titer Glo, Flow Cytometry
Target Cell Type MDA-MB-231 adherent and K562 suspension cells
Experiment Plan Scan plate using Far Red and Bright field channels
Hypothesis Drug treatment will increase the percentage of DRAQ7-positive cells over time

Celigo Setup

Plate Type Greiner cat# 781091 384-well black wall clear bottom
Scan Channels Far Red, Bright field
Resolution 1 µm/pixel
Scan Area Whole well
Analysis Method Target 1 + 2
Scan Frequency Daily, for 3 days
Scan Time ~15 minutes

Assay Protocol and Plate Setup

Goal:

Detect and quantify dead cells using DRAQ7 stain in adherent MDA-MB-231 and suspension K562 cell lines

Protocol:

  • Seeded MDA-MB-231 at 2,000 cells/well and allowed to incubate overnight
  • Suspension cells were plated the day of experiment with a working solution of DRAQ7 at 3,000 cells/well
  • Prepared the drug Benzethonium and serially diluted to generate dose response
  • Prepared control with water in media
  • Removed the media from the wells of adherent cells
  • Added drug dose response and control to both adherent and suspension cells
  • Added DRAQ7 to adherent cells only
  • Incubated the plate for 24, 48 and 72 hours with drug and dye
  • Imaged the plate using the Celigo image cytometer

Plate map for Benzethonium (µM) drug treatment and DRAQ7 time point staining

kinetic viability DRAQ7 plate set up

Results

Drug-treated MDA-MB-231 and K562 cells showed an increase in DRAQ7-positive cells

  • DRAQ7-positive cells were determined by staining the cells for 24, 48 and 72 hours

Images and fluorescent object identification looked as shown below for DRAQ7-stained cells “Graphic Overlay” segmentation

kinetic viability cell images

Results for the MDA-MB-31 and K562 counts of dead cells after 24 hours of Benzethonium drug treatment

kinetic viability plate level data

Graphs

1. Graphs were generated using Graph Pad Prism for the dose-response of Benzethonium after 24, 48 and 72 hours treatment. In this experiment, the average of 4 data points was plotted.
2. IC50 values were calculated using Graph Pad Prism

  • Cell death increased over time with Benzethonium (14.5 µM) versus the control

kinetic viability cell death graphs

Conclusion

  • Drug-treated MDA-MB-231 and K562 cell lines were successfully imaged and analyzed on Celigo
  • Kinetic viability assay using DRAQ7 allows for the enumeration of total counts of DRAQ7-positive cells over the time
  • Acquisition of high-resolution DRAQ7 and bright field images of 384-well plate took about 15 minutes
2018-10-11T19:04:47+00:00