Flow cytometric analysis of human,,,primary cells using the Agilent 2100,,,bioanalyzer and on-chip staining
for handling few cellsOn-chip CBNF staining and stepsfor handling few cells aredescribed in reference 2 usingprocedure B. Experiments and Results We have developed tools for measuringthe transfection efficiencyusing a GFP reporter plasmid inprimary cells on the 2100 bioanalyzer.Great emphasis was placedon being able to
for handling few cells
On-chip CBNF staining and steps
for handling few cells are
described in reference 2, using
procedure B.
Experiments and Results
We have developed tools for measuring
the transfection efficiency
using a GFP reporter plasmid in
primary cells on the 2100 bioanalyzer.
Great emphasis was placed
on being able to perform the
analysis with a minimum of cells
and to eliminate steps in the protocol
where cells may be lost.
These protocols facilitate working with very few cells. A concentrated
cell buffer (2x CB) was
added to the Cell Fluorescence
Lab Chip kit. This buffer allows
the exact adjustment of cell concentration
and density of samples
with few cells. Cells were transfected
and stained on-chip with
the live cell stain CBNF as
described in reference 2. Only
30,000 cells were required for onchip
staining of primary cells and subsequent measurement on the
2100 bioanalyzer. Figure 1 shows
an optimization study conducted
to reach best transfection efficiency
in HUVEC cells. HUVEC cells
were transfected with decreasing
DNA:Lipofectamine (LP) ratios
(1:1 to 1:10). Figure 1A shows the
transfected samples as viewed
through a fluorescence microscope
18 hours after transfection
and figure 1B shows the corre-
sponding dot plots generated on
the 2100 bioanalyzer. The selected
region shows the positively transfected
live cells. Figure 1C shows
the GFP-histograms of the samples
with the percentages of double
positive cells. The recovery of
live cells, as determined by manual
counting under microscope
after trypan blue straining,
decreased as the amount of Lipofectamine
was increased (compare listed percentages of recovery
in figure 1A) due to the toxic
effect of Lipofectamine on the
cells. However, the transfection
efficiency steadily increased up to
a DNA:Li
'"/>Source:
Page: All 1 2 3 Related biology technology :1.
High-throughput gene expression analysis to screen for anti-asthma drugs2.
Fully automated DNA purification and efficient multiplex PCR for analysis
of microsatellite loci3.
2100 expert software
Powerful software for the analysis of RNA, DNA, proteins, and cells with the Agilent 2100 bioanalyzer4.
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