Measurement of Differential Apoptosis of HEK cells in presence of PDGF

January 06, 2015

George Washington University, Washington DC
Surgery
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

Annexin V FITC conjugated

Catalog Number:

640906

Apoptosis of keratinocytes is a crucial step that balances proliferation to maintain proper epidermal thickness. However keratinocytes from the peripheral area of chronic wounds undergo higher and abnormal apoptosis which hinders successful re-establishment of the epidermal layer in order to achieve the desired wound healing. We focus on the effects of growth factor and cytokines on apoptosis of keratinocyte in presence of an apoptosis inducer. We found that distinct growth factors and small molecules could affect the apoptosis of keratinocytes and thus may be considered as potential targets to be used as therapeutic agents in chronic wounds.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

HEK cells

Protocol Overview

Human Epidermal Keratinocyte (HEK cells) were either left untreated, kept under different concentrations of H2O2 for four hours, or kept with the highest concentration of H2O2 with PFGF. Apoptosis was measured by staining for Annexin V according to the manufacturer’s protocol, followed by flow cytometry. Briefly 1X10 5 cells were stained in a final volume of 100ul with 2.5 ul of Annexin V (FITC). Incubation was done for 20 minutes in ice followed by addition of another 100ul of 7AAD in PBS (in the dilution of 1:2000). The final volume should now be 200ul sufficient to be assessed by flow cytometry.

Tips

Concentration and time of exposure to Annexin and 7AAD is crucial. Turn on FACS before adding the antibody or the dye. Be sure that intermittent steps do not delay the start of cell acquisition in Flow cytometer.

Results Summary

Flow cytometric method of analysis of apoptosis was employed in HEK cells that were either untreated or exposed to different concentration of hydrogen peroxide and in another group which is exposed to the highest concentration of hydrogen peroxide along with PDGF (0.5ng/ml). As expected, there was significant increase in apoptosis of HEK cells as the concentration of hydrogen peroxide exposure increased. But apoptosis was reduced at the highest concentration of hydrogen peroxide when PDGF was added. This indicated the PDGF had a key role in inhibiting or at least slowing down induced apoptosis in HEK cells

Additional Notes

Incubation time in Annexin V (FITC) may vary with different cell type.

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Summary

The Good

Good staining. Clear differences between live and apoptotic cells. Easy protocol. Entire experiment takes about 1-2 hours including cell acquistion.

The Bad

None

The Bottom Line

Good, easy method of analysis of apoptosis. Experiment cost is cheap but reliable and repeatable.

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