Wednesday, March 12

CFSE labelling of heat killed Bps

I know I probably should have "asked" you all if anyone had advice before I started my experiment today, but I've been rather busy "chatting" with Miss A via email, cleaning the lab, doing vaccinations for Tash, doing category 3 harvests for Tash, and setting up stimulation experiments for myself, so suddenly I remembered I wanted to do this experiment, so I looked up a protocol from a paper I'd recently read (Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils. Tuominen-Gustafsson et al, BMC Microbiology October 2006). So my point which is somewhat non-valid anymore as in a few hours I should have the answer for myself, but let's have a mini-contest to see who can guess the if it's going to work or not (obviously if you have a science background & can give me more validation as to why you think it will or will not work that would be wonderful as well!)

I would like to label some heat killed Bps with CFSE & then see if I can see them on the FACS machine. I think it would be REALLY cool to then give these bugs in & then look to see where they go &which cells they're interacting with, the problem is I'm not sure that dead Bps will pick up the CFSE stain in the first place. I'm not worried about the fact that they're dead & won't divide, I understand that, just wonder if my morning of staining is going to work or not in the first place. It's a very crude experiment & now I'm just waiting for the FACS machine to be free.

So my question to all of my readers (all 3 of you) do you reckon this will work & why????

UPDATE 18.15: I know the results so please give a guess & I'll let you know tomorrow.

Also Laura & I just booked our tickets to Berlin for a TB symposium in June, we're so excited to get a day of sight seeing on Saturday as well!!

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At 12 March, 2008 23:04, Blogger PCS said...

It should work as long as the heat treatment of the bacteria doesn't denature the DNA. I'd try it a couple of ways. CSFE treat heat killed bacteria, CSFE treat live bacteria then kill them.

You could also treat heat killed bacteria with immune serum then label them with a labeled anti-antibody. I know that should work because that is how we used to do Toxo.

At 13 March, 2008 11:52, Blogger Sara said...

well as I guessed PCS knew the correct answer!

I want to try CFSE labelling the live bacteria, but since there is no cat 3 protocol, I had to try it out first in cat 2 & now have to write up the protocol, regulations in the UK are very different from the US, in my mind more annoying, but safety IS always the first priority & I never disagree with that, just when you get excited about an experiment & then have to start out this slower method I get a bit anxious.

Off to write up the protocol to be reviewed by the cat 3 safety officer now :)

At 15 March, 2008 04:09, Anonymous Anonymous said...

OK, I'm impressed but mostly confused. Just stopped by to say hi.

Love, Mommio

At 08 August, 2012 07:23, Anonymous CSFE labelling said...

It should work as long as the CSFE heat treatment of the bacteria doesn't denature the DNA.


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