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 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 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:Lipofectamine ratio of 1:8.
When more Lipofectamine was
used transfection efficiency
decreased again. This data shows
that to obtain a maximum number
of live transfected cells it is important
to take the toxicity of the transfection reagent into account.
For this experiment, a ratio of 1:6
was found to be optimal. In those
cases where enough cells were
obtained, samples were measured
in parallel on a flow cytometer
(figure 1D - only GFP histograms
are shown). We also determined
the optimal transfection condition
for NHDF using a similar protocol.
Figure 2 shows the results
obtained when the cells were transfected with the optimal
DNA:Lipofectamine ratio of 1:8.
Figure 2 represents the dot plots
of the control (A), the transfected
sample (B) and the GFP histograms
- control (C) and transfected
(D). Although on average
only 650 events were counted per
sample data quality was very good
for the histograms and dot plots.
Figure 2E shows the untransfected
and figure 2G the transfected
samples viewed using a fluorescence
microscope 18 hours after
transfection. Figure 2F is a phase
contrast photograph of the control
cells.
|
|
Conclusion
|
Primary cells are valuable samples
and are frequently limited in availability
and lifespan. It can therefore
be difficult to conduct experiments
with these cells. The
Agilent 2100 bioanalyzer with its
low sample consumption together
with an on-chip staining protocol
proves to be a versatile tool for
analysis of primary cells. The complete
staining procedure takes
place on chip and the analysis
requires minimal amounts of cells
and reagents, saving both time and
reducing sample consumption.
|