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MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death
Norbert Blank1, Renate Burger, Birgit Duerr
1Institute for Clinical Immunology, Department of Medicine III, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
Background:
Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126.
Methods:
Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining.
Results:
U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses.
Conclusions:
These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered.
Insights
The MEK inhibitor U0126 causes autofluorescence in cells, interfering with apoptosis detection via Annexin V/Propidium Iodide staining. Researchers recommend using alternative MEK inhibitors or non-fluorescent methods for accurate apoptosis assessment.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Mitogen-activated protein kinase (MAPK) signaling is crucial for cell survival and proliferation.
- MEK (MAPK kinase) regulates MAPK activity; MEK inhibition or growth factor deprivation induces apoptosis.
- Apoptosis detection commonly uses phosphatidylserine (PS) exposure and propidium iodide (PI) exclusion via flow cytometry.
Purpose of the Study:
- To investigate the limitations of detecting apoptotic cell death using cytofluorometry in cells treated with the MEK inhibitor U0126.
- To understand how U0126 affects fluorescence-based apoptosis assays.
Main Methods:
- Apoptotic cell death was induced in INA-6 cells, peripheral blood mononuclear cells (PBMC), and T lymphoblasts.
- Induction methods included interleukin-6 (IL-6) deprivation or incubation with U0126.
- Apoptosis was quantified using Annexin V/Propidium Iodide (AxV/PI) double staining via flow cytometry.
Main Results:
- U0126 treatment led to significant changes in cellular fluorescence patterns.
- Background fluorescence in U0126-treated cells mimicked PS exposure on viable cells, complicating AxV/PI staining.
- U0126 developed intrinsic fluorescence during cell culture, detectable across fluorescence channels, particularly FL1 (525 nm).
- This U0126-induced fluorescence was partly dependent on cellular uptake and intracellular modifications.
Conclusions:
- Appropriate controls are essential for all time points in fluorescence analyses involving chemical enzyme inhibitors.
- For MEK inhibitors, consider alternatives like PD098059 or PD184352.
- Non-fluorometric methods for apoptosis detection should be considered when using U0126.