additional histogram peak between G1 and G2

Greetings, when using EtOH fixation of human fibroblasts, I am 
occasionally getting DNA histograms (after PI/RNase staining) that show 
a peak somewhere in between G1 and G2, which I suppose could be due to a 
minority of cells whose DNA staining (or fixation perhaps) went 
differently from the others. That's quite critical because I need to 
estimate S-phase fractions (using a model) in my BrdU negative 
populations (you'll see it in the attached graphs)

This is what I do (got it from my friend Chang Uk Lim in Colorado State 
Uni):
1) Wash pellet in PBS, re-suspend approximately 500,000 cells in 0.5ml 
of PBS
2) Add drop wise, while vortexing at a mid-speed, 1.0ml 0f 100% EtOH 
that had been stored at -20C. Then I add another 250ul, making it 
gradually to 70%EtOH.
3) Store cells at 4C, then wash pellets in 1% BSA on the day when 
treatment for immunofluorescence is due
4) Expose to HCl 2N supplemented with 0.2mg/ml pepsin for 15 minutes at 
37degC (Working volume is 350uL, this is in preparation of 
immunodetecion of BrdU with MAb)
5) Stop hydrolysis with 1mM Tris pH10, 0.94ml
6) Spin and Wash with 1%BSA
7) Carry on with primary and secondaries for BrdU,using Tween and BSA. 
Rat primary anti BrdU (AbCam) and Alexa 633 goat anti rat.
8) Stain DNA with PI and RNase, don't have details with me (they're in 
the lab book in the lab) but really this has always gone very well 
otherwise.
9) Get on a BD FACS Calibur with Cell Quest.

I have tried to increase my vortexing speed a bit, since the first time 
that I had run this protocol I did notice that EtOH and H2O weren't 
quite mixing well in my tube. Things have possibly improved a little 
bit, but I wonder whether I am doing anything else wrong. There may be a 
margin for increasing my vortexing speed a bit more.

A few examples are attached. There was no compensation set between FL2 
and FL4. The plots are produced using FlowJo for Windows. 150,000 events 
were collected.
The pdf files are mid-vortexing speed
The jpg files were my first run, were I was being perhaps too kind with 
my vortexing.

Any comment would be most welcome,

Cheers
Massimo

-- 
Massimo Pinto
Post-doctoral fellow
Radiation Research, Radiology
MSB-F451, NJMS, UMDNJ
185 South Orange Avenue
Newark, NJ 07103-2714, U.S.A.
ph. (+1)973-972-6961
massimo.pinto@umdnj.edu


This attachment - 'pseudocolours-odd-population.pdf' -	64.76 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/4c4fa1580544076488be1225c9b6607fa884cbda.pdf 


This attachment - 'histogram-odd-population.pdf' -  6.45 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/38c324a7930ae779e04eed836b496bba789a118d.pdf 


This attachment - 'odd-population-pseudocolour.jpg' -  40.78 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/cdb2424cfd7c5148d151becbda8b7fd18984a482.jpg 


This attachment - 'BrdU negatives-odd-peak.jpg' -  21.83 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/6e8f85dea02d8c1087af0a969a7a22e7b9a7b1b9.jpg