4 replies to this topic

[SuMi]

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.

[laurequillo]

You could try DMEM without phenol red...

[virusfan]

Could it be autofluorescence?

[SuMi]

virusfan, on 17 September 2010 - 07:49 AM, 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

[BNodes]

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 23 September 2010 - 03:38 AM, said:

virusfan, on 17 September 2010 - 07:49 AM, 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