Hi everyone, I am analysing cell turnover in
HIV infection using staining with Ki67 antibody. We first made a series of experiments
using different permeabilizing methods: FACS lysing solution, EtOH, and Cytofix/cytoperm
from parmingen. We decide to adopt the EtOH (70%, 30 min at -20 degrees) method. Now
that we are analysing patientīs samples I have the feeling that the method is not working
properly. First a lot of events donīt fall in the lymphocyte gate, but out of scale (cell
clumps?, dead cells?, etc..), so we can only acquire few lymphocytes events. Second
we are founding no staining at all in the majority of samples, even in untreated
patients with high plasma viremia where one expect to find a high proportion of Ki67+
T cells. So could you please comment on this with your personal experiences on whivch
is the best method to permeabilise, etc... The Ab we are using is from B&D.
Many thanks
Jose Miguel Benito
Hospital Carlos III
Madrid
Spain