RE: SIMULTANEOUS SURFACE AND PI STAINING QUESTION

Dear Ioannis,
Concerning your questions:
1.I never performed ethanol fixation after APC staining, but ethanol might
affect APC, just like it affects R-PE, published 12 years ago by Ken Toba in
Cytometry 1992;13(1):60-7. "Improved staining method for the simultaneous
flow cytofluorometric analysis of DNA content, S-phase fraction, and surface
phenotype using single laser instrumentation." We observed exactly the same.
2. It is no problem using log amps for FITC and APC.
3. It might be possible that you are dealing with doublets. For a more
optimal discrimination between singlets and doublets using PI and APC as
fluorescent dyes for labelling DNA and a protein, respectively, you need to
change the hardware of the Calibur, since APC is measured on the FL4 at the
costs of the pulse-width (FL1, FL2 or FL3). The solution is quite simple.
You only need a new connecting wire (can be obtained from BD) and make some
disconnections and new connections in the Calibur. The protocol was
published in the following paper: "Four-color multiparameter DNA flow
cytometric method to study phenotypic intratumor heterogeneity in cervical
cancer.
Cytometry. 2000 Feb 1;39(2):96-107."
Good luck,
Willem Corver




-----Original Message-----
From: Kotsianidis, Ioannis [mailto:i.kotsianidis@imperial.ac.uk]
Sent: maandag 20 december 2004 16:03
To: cyto-inbox
Subject: SIMULTANEOUS SURFACE AND PI STAINING QUESTION



Dear flowers,


I have a couple of questions regarding simultaneous surface  and cell cycle
analysis with PI.


I stained a lymphoblastoid cell line with FITC and APC conjugates,
subsequently

fixed it with 70% ethanol and then incubated it with RNAse and PI.

I acquired the cells in a FacsCalibur (settings set for cell cycle analysis,
PI is read on FL2-A) and I saw 

a strange picture of double positive events at the FL1 vs FL4 plot. 

I repeated the acquisition with my usual four color settings 

and I've noticed that there was no positivity for APC, while my unfixed
sample was

all double positive for both FITC and APC.


1) Does the fixation destroyed the APC epitope (if yes, then why not also
the FITC)?


2) Can I use FL4 and FL1 in Log scale for acquisition and analysis, while I
perform cell cycle analysis with PI?


Thanks in advance,




Dr Ioannis Kotsianidis 
Clinical Research Fellow 
Department of Haematology 
Faculty of Medicine - ICSTM 
Hammersmith Hospital 
Du Cane Road 
LONDON W12 0NN 
U.K.