RE: Protein Content

Hi Ultan-
Thanks for your detailed response.  I failed to clarify that I was looking
to quantify protein content "within" the cell cycle, i.e. G0/G1, S, G2/M.
I'm aware of the uses of CFSE for tracking cell divisions; unfortunately,
I'm unaware of how to properly phrase a question to the Purdue list! 
I know that Pyronin Y is used for this application.  I'm simply wondering,
based on the huge availability of fluorescent dyes as succinimidyl esters,
whether anybody had tried to duplicate early efforts to quantify protein
using isothiocyanates like FITC (as per the paper I noted).
Thanks again for your post.  I know that generating detailed explanations
like this can interrupt one's workflow, but the benefit to the flow
community is huge.
Have a wonderful Christmas.
Andrew

  _____  

From: Ultan Cronin [mailto:ultan.cronin@ul.ie] 
Sent: Thursday, December 21, 2006 1:04 AM
To: cyto-inbox
Subject: Re: Protein Content


Hi Andrew,

Recently I used CFDA-SE (it's sometimes called CFSE) to monitor cell
division in batch cultures of a bacterium, Bacillus cereus.  The idea was to
stain inoculum cells with CFSE and, as cells divided during batch culutre,
daughter cells would have 50% of the fluorescence of mother cells.  Over
time the mean fluorescence of the population would decrease and I had hoped
that different peaks would be evident  - e.g. cells that had undergone 8
divisions compared with those that had undergone 12.  Over the course of the
batch culture, the mean fluorescence of the cells did decrease.  However
only one broad peak could be seen at any given sample time - no specific
peaks for cells resulting from a certain number of divisions were
discernable.  One interesting finding is that the CV of the peak decreased
over time, which we interpreted as meaning that heterogeneity in terms of
cell proten content decreased as the batch culture progressed. 
I would say that CFSE was a good marker for protein content for my species.
Fluorescence was maintained even after six days of culture meaning that the
probe really suck on well to proteins.	We kept the culutures in the dark so
no photobleaching occurred.  We did not take steps to prevent oxidation of
the fluorochrome, but over six days this did not seem to be a problem.

A couple of papers I found very insightful were:
Hoefel D, Grooby WL, Monis PT, Andrews S, Saint CP. A comparative study of
carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl
ester as indicators of bacterial activity. Journal of Microbiological
Methods 2003;52(3):379-388.
Lyons AB. Divided we stand: tracking cell proliferation with
carboxyfluorescein diacetate succinimidyl ester. Immunology and Cell Biology
1999;77:509-515.

I know all the above concerns bacteia but I hope I have been of some help.

Ultan



  _____  

Ultan Cronin BSc MSc
Dept of Life Sciences
Schrodinger Building
University of Limerick
Castletroy
Co. Limerick
Ireland
Tel: 00353-(0)61202890
Mob: 00353-(0)872032963
Fax: 00353-(0)61331490