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I was told by the Invitrogen technical team that the Organelle Light was 
working on hippocampal neurons in culture, and that it has been tested 
in-house by Invitrogen.

This protocol seems really harsh, so I'm wondering if it's the same for 
neurons ? Particularily, the "quiescent cells will not be infected" part 
and the room temperature incubation.

Can you confirm that Organelle Light can be used in neurons, and give me 
the appropriate protocol ?

Christophe Leterrier

Johnson, Iain a écrit :
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> **Manufacturer Response**
>  
> The following is from Rob Batchelor, who did much of the development
> work on the Organelle Lights:
>  
> In my hands CHO cells work well (60%+ transduction).  I would suggest
> the following:
>  
> 1.  Plate cells so they are 60-75% confluent when you add the virus.
> 2.  Dilute the virus supplied 2 ml to 3.5 ml PBS without Ca++ or Mg++
> (very important; divalent cations seem to depress virus entry).  You can
> change the volumes to whatever you need for your plate/dish set up, but
> keep the ratio the same.
> 3.  Rinse the cells once with PBS without Ca++ or Mg++
> 4.  Add diluted virus to cells.
> 5.  Incubate in the dark at room temp (20-25C) for 2-4 hours.  37C
> incubation will result in _no_ transduction.
> 6.  Aspirate virus.
> 7.  Add 1X enhancer in the appropriate media plus serum.
> 8.  Incubate for 90-120 minutes at 37C, 5% CO2.
> 9.  Aspirate media+enhancer, add back the appropriate media plus serum
> 10. Incubate overnight at 37C, 5% CO2.
> 11. Image.
>  
> I haven't tried HEPES buffer, but I don't see why it wouldn't work.  
> Media has Ca++ and Mg++, obviously; I wouldn't suggest that.
>  
> If you have any questions or comments, please email me directly:
> [log in to unmask] <mailto:[log in to unmask]>
>  
> I would further emphasize that the incubation with virus at ROOM
> TEMPERATURE NOT 37C is particularly important (I have made this mistake
> myself and learned the hard way.....).  Also the the confluence of the
> culture (# 1, above) is key.  You want the cells to be in log phase
> growth if possible. Quiescent cultures will not work.
>  
> Iain
>  
>  
>
> ------------------------------------------------------------------------
> *From:* Confocal Microscopy List [mailto:[log in to unmask]]
> *On Behalf Of *Claire Brown
> *Sent:* Thursday, July 12, 2007 11:08 AM
> *To:* [log in to unmask]
> *Subject:* Organelle Lights
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I am going to test out the Molecular Probes Cellular Lights on my CHO
> cells and I was wondering if anyone has any tips on getting this to
> work. I have a co-worker who was having trouble getting it to work and I
> thought I would see if others have made any changes to the protocol and
> had good success. I am a little concerned about the step to put the
> cells in PBS at room temp for 2-4 hours. I was thinking of working in
> HEPES buffered serum free media and maybe putting the cells at 37.
>
>  
>
> Any advice would be appreciated.
>
>  
>
> Sincerely,
>
>  
>
> Claire
>
>  
>
> /_________________________________________________________________/
>
> /Claire M. Brown, PhD/
>
> /Life Sciences Complex Imaging Facility Director/
>
> /McGill University Department of Biochemistry/
>
> /**http://www.lifesciencescomplex.mcgill.ca/imaging**
> <http://www.lifesciencescomplex.mcgill.ca>/
>
>  
>