Anyone heard of cells spontaneously fluorescing red?? - (Sep/17/2010 )
I had a very weird experience yesterday. I was running my Raji B cells on the flow cytometer and there was a positive red fluorescent peak in my negative sample. I went back to my flask of cells and ran an aliquot through the flow and they too were positive for red fluorescence. I looked at the cells under the microscope and some of the cells are glowing red. These cells weren't labelled with anything and so I have no idea as to how this spontaneous fluorescing happened. Anyone have any ideas? The only explanation I have is that it could be something in the media causing this but I have never seen it before and my cells were washed and resuspended in PBS before I ran them on the flow.
You could try DMEM without phenol red...
Could it be autofluorescence?
virusfan on Fri Sep 17 15:49:09 2010 said:
Could it be autofluorescence?
I thought of that but dismissed it as cells would normally autofluoresce in the green channel and this was the red channel. There was no fluorescence in the green channel. I could try growing them without phenol red but I have been using these cells for two years and I've never had this problem before so I can't see how the medium could be the problem
Cells autofluoresce in all channels. More importantly, I've noticed that when cells are stressed or when they apoptose the produce some fluorescent red chemical/protein. It should be relatively faint, and you should be able to separate them out with FSC and SSC gates.
-Bill
SuMi on Thu Sep 23 11:38:53 2010 said:
virusfan on Fri Sep 17 15:49:09 2010 said:
Could it be autofluorescence?
I thought of that but dismissed it as cells would normally autofluoresce in the green channel and this was the red channel. There was no fluorescence in the green channel. I could try growing them without phenol red but I have been using these cells for two years and I've never had this problem before so I can't see how the medium could be the problem