57
OCTOBER 16-18, 2011 • PORTLAND, OR
P21
No-Lyse No-Wash Immunophenotyping Using
Acoustic Cytometry
Rick Kerndt, Jolene A Bradford
Flow Cytometry Systems, Life Technologies, Eugene,
OR, USA
Immunophenotyping whole blood is a primary
application in the study of white blood cell populations
and their function. Red blood cells (RBCs) traditionally
have been removed during the preparation step by
lysis methods before or after antibody staining, or by
removal using density-gradient selection followed by
washing steps to remove red blood cell fragments
and platelets. This processing of whole blood has
its draw backs due to the potential loss of cells of
interest and the diffculty in automating cell washing.
Methods have been previously described using a
no-lyse, no-wash staining protocol with the sacrifce
of light scatter resolution. Accurate identifcation of
some cell populations depends on high-resolution
scatter data, so previous no-lyse, no-wash protocols
require high sample dilutions to reduce coincidence
with red blood cells. We present a no-lyse, no-wash
method which takes advantage of acoustic focusing
technology to provide, fast, sensitive and accurate
measurements of dilute samples. A fve color panel
is presented which uses a membrane permeable
nucleic acid dye (Vybrant® DyeCycle™ Ruby stain
) to identify nucleated cells from the whole blood
core stream. The remaining panel comprises a basic
immunophenotyping using mouse anti-Human CD19,
CD3, CD4, CD8, and CD56. A total of 10 mL whole
blood is stained in a 50 mL labeling volume and then
diluted 1:400 fold (4ml) in PBS prior to acquisition
on an acoustic focusing cytometer. Light scatter
properties and sub-population percentages are
equivalent to lyse wash immunophenotyping protocols
with comparable acquisition times.
POSTER ABSTRACTS