Flow cytometric analysis of human,,,primary cells using the Agilent 2100,,,bioanalyzer and on-chip staining
A fast and accurate method to detect green fluorescent proteinexpression in samples of low cell numbers This Application Noteaddresses the major problemsthat many researchers workingwith primary cells have for examplelimited availability quant
A fast and accurate method to detect green fluorescent protein
expression in samples of low cell numbers
This Application Note
addresses the major problems
that many researchers working
with primary cells have, for example,
limited availability, quantity
and lifespan. Using the Agilent
2100 bioanalyzer and Cell Fluorescence
LabChip kit the transfection
efficiency of primary cells
can be determined using green
fluorescent protein (GFP) as a
reporter molecule. Two types of cells were
transfected and analyzed: human
umbilical vein endothelial cells
(HUVEC) and normal human dermal
fibroblasts (NHDF). The
advantages of using the compact
2100 bioanalyzer for monitoring
transfection efficiency in primary
cells include low cell consumption,
high reproducibility of
results, fast on-chip staining
procedure and easy-of-use.
Materials and Methods
Primary cell culture
HUVEC, NHDF, culture media, and
trypsin/EDTA solution were
obtained from Clonetics. HUVEC
cells were maintained in EGM-2
medium and NHDF cells were
cultured in FGM-2 medium.
Transfection
Cells were trypsinized and seeded
into 12-well culture plates at a
density of 3 105 cells/mL in 1 mL
of culture medium and incubated
for 20 hours under cell culture
conditions. Prior to transfection
the growth medium was replaced
by 1 mL of OPTI-MEM I (Gibco).
DNA-Lipofectamine 2000 (Invitrogen)
complex was prepared and
added to the cells according to the
supplier's protocol. After six
hours of incubation, the transfection
medium was removed and
replaced with 1 mL of culture
medium. The cells were harvested
18 hours later.
On-chip CBNF staining and
steps
'"/>Source:
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