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Home  |  Protocols  |  In situ hybridization and Immunochemistry  |  Labelling authophagosomes in live cells

Protocol - Labelling authophagosomes in live cells

Labelling authophagosomes in HaCaT live cells with monodansylcadaverine, after UVB stimulation

CategoryIn situ hybridization and Immunochemistry
Last revisionJan 29, 2015
Author(s)Kisslinger A, Paladino S.,Tramontano D.
Contact
NameAnnamaria Kisslinger
AddressInstitute of Experimental Endocrinology and Oncology “G. Salvatore” , CNR via Pansini 5, 80131 Naples Italy
Phone+3908107464552
Emaila.kisslinger@ieos.cnr.it

 

Figure legend:

Monodansylcadaverin staining of HaCaT cells after UVB stimulation


Steps

Description Temperature Time Note
Seed cells on bottom-glass dishes
37°C, 5% CO2, in a humidified incubator
24 hrs before experiment
Experimental culture conditions: semiconfluentt
Wash with PBS buffer three times
RT
Irradiate with UVB source
RT
10 - 100mJ/cm2
without lid
Add monodansylcadaverine 50µM in PBS buffer
37°C, 5% CO2, in a humidified incubator
10 min
Collect images
RT
immediately after incubation

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.

 

Quality validation: Yes

Validation info

The protocol has been published in a peer-reviewed journal

 

Citations
Vitale N, Kisslinger A, Paladino S, Procaccini C, Matarese G, Pierantoni GM, Mancini FP, Tramontano D. Resveratrol couples apoptosis with autophagy in UVB-irradiated HaCaT cells PLoS One. 2013 Nov 19;8(11):e80728. doi: 10.1371

 

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