Protocol - Labelling authophagosomes in live cells
Labelling authophagosomes in HaCaT live cells with monodansylcadaverine, after UVB stimulation
Figure legend:
Monodansylcadaverin staining of HaCaT cells after UVB stimulation
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Seed cells on bottom-glass dishes
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37°C, 5% CO2, in a humidified incubator
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24 hrs before experiment
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Experimental culture conditions: semiconfluentt
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Wash with PBS buffer three times
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RT
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||
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Irradiate with UVB source
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RT
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10 - 100mJ/cm2
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without lid
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Add monodansylcadaverine 50µM in PBS buffer
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37°C, 5% CO2, in a humidified incubator
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10 min
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|
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Collect images
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RT
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immediately after incubation
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Other informations
The irradiating source consists of three lamps (Philips Ultraviolet 8 TL 20W/01 RS lamps; Philips, Eindhoven, Netherlands) generating UVB light in the range of 290–320 nm with an emission peak at 312 nm. Intensity of UVB irradiation was measured using a phototherapy radiometer (International Light, Newburyport, MA).
Images were collected using a laser scanning microscope (LSM 510 META, Carl Zeiss Microimaging, Inc.) equipped with a planapo 63x oil-immersion (NA 1.4) objective lens. All image processing was done using LSM 510 software.
Validation info
The protocol has been published in a peer-reviewed journal
