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.