| Comparison of the
standard and the alternative protocol:
Besides the standard protocol, we
developed an alternative protocol and tested several
cell lines for their receptivity of the antibody. With both
procedures, cells were plated
at a density of approximately 30 thousand cells/well in a
48-well plate one day before
protein transfection. Cells were confluent the next day.
Transfection complexes were prepared
consisting of 10µL SAINT-PhD reagent, the protein of interest,
and HBS buffer as diluent in
a total volume of 25µL/well. Protein complex formation occurred
immediately and an additional
100µL of total growth medium (supplemented with 10% FBS) ) was
added if the standard delivery
procedure with SAINT-PhD was used. Culture medium was aspirated
from the cells and the prepared
complex (125µL) was added to each well. The cells were incubated
with the mixture for 4 hours
(37°C, 5% CO2) and then harvested by trypsinization. The FITC
conjugated antibody was detected
by FACS analysis. As negative controls the SAINT-PhD and the
antibody alone (not complexed together)
was added to the cells and last-mentioned showed less than 1%
FITC positive (data not shown).
Standard protocol:
Using the standard delivery procedure we delivered various
quantities of a FITC-labeled antibody
with SAINT-PhD. Complexes were added to CHO-K1, 3T3 or COS-7
cells (80% confluent) in total growth
medium and followed by 4 hours of incubation (37°C, 5% CO2). No
visible cell death occurred during
this time. As shown in figure 1, intracellular delivery of the
antibody was already observed after
4 hours incubation and FACS analysis shows high percentages of
cells containing the antibody.
Data are presented as histograms of the
percentage fluorescent cells for each cell line. Gates were
set to distinguish between auto fluorescence of control cells
and that of highly fluorescent cells.
The efficiency of delivery is clearly dependent on the amount of
protein used.
Figure 1: Intracellular delivery of FITC-labeled IgG with
SAINT-PhD: A range of FITC-labeled
IgG was mixed with 10µL of SAINT-PhD reagent in a total volume
of 25µL. The complexes were filled
up to 125µL with total growth medium and added to the cells in a
48-well plate. The complexes were
incubated for 4 hours (37°C,5% CO2) without changing the medium.
FACS analysis was performed
after harvesting the cells.
SAINT-PhD is the only serum compatible
protein delivery reagent. So this method of protein
delivery works fast and efficient without the need of changing
the medium. Within a few hours
you can perform your assay.
Alternative protocol:
Because transfection by SAINT-PhD is not influenced by total
growth medium (containing 10% FBS)
we developed an even faster method, involving less hands-on
time. Therefore the same experiment
as above was performed, but instead of replacing the medium with
a complex-containing-medium,
we added the 25µL antibody/HBS/SAINT-PhD complex drop wise to
each well with cells and incubated
at 37°C and 5% CO2 for 4 hours. Cells were harvested and
analyzed by FACS (figure 2). Results
were compared with the standard delivery method.
Figure 2: Intracellular delivery of FITC-labeled IgG with
SAINT-PhD: A range of FITC-labeled IgG
was mixed with 10µL of SAINT-PhD reagent in a total volume of
25µL. The complexes were added
directly to the cells in a 48-well plate. The complexes were
incubated for 4 hours (37°C,5% CO2)
without changing the medium. After 4 hours cells were harvested
and analyzed by FACS.
This experiment shows that protein
delivery mediated by SAINT-PhD is dependent on the culture
volume in which the cells are growing. Withthe alternative
delivery method the cells are in their
original seeding volume of 350µL per well. So, with the complex
added, the end-volume is 375µL.
In the standard delivery procedure the end-volume is 125µL,
which is a three-fold less volume. As
illustrated in figure 2, a 3-fold increase in the end-volume
reduces the level of fluorescent signal
observed in the cell lines after 4 hours of incubation. Only
CHO-K1 seems not to be influenced by
the delivery capacity in a larger end-volume. Because the
concentration of the transfection complex
is lower in the larger volume, the speed of delivery will be
slower, therefore we also looked at the
delivery of the antibody after 24 hours of incubation (figure
3).
Figure 3: Intracellular delivery of
FITC-labeled IgG with SAINT-PhD: A range of FITC-labeled IgG
was mixed with 10µL of SAINT-PhD reagent in a total volume of
25µL. The complexes were added directly
to the cells in a 48-well plate. The complexes were incubated
for 24 hours (37°C,5% CO2) without
changing the medium. After 24 hours cells were harvested and
analyzed by FACS. From figure 3 it is clear that after 24 hours
of incubation very high levels of delivery are reached
(up to 97% for CHO K1).
Summary
Two protocols have been developed: the standard protocol and an
alternative protocol. Above we have
shown the results for the delivery of F(ab’)2 Rabbit-anti-Mouse
IgG-FITC with SAINT-PhD in three
different cell lines: CHO-K1; COS-7 and 3T3. With the standard
protocol, a little bit more
hands-on-time and some more pipetting is involved. However, with
the standard protocol already after
4 hours high levels of delivered protein can be observed. In
contrast, the alternative protocol
involves much less pipetting and less hands-on-time. A minor
matter is that in case of the
alternative protocol it takes 24 hours of incubation to reach a
high level of intracellular delivery
ofthe protein. Both protocols can be used to efficiently deliver
relatively large amounts of a
protein inside a cell.
However, it is up to the person performing
the experiments to choose the most convenient protocol
with respect to time and resources available. Furthermore, other
cell lines or primary cells might
benefit more from one or the other protocol. The two protocols
presented above are merely a starting
point for our customers to begin their experiments with. The
best results always are obtained after
optimization of a protocol for a specific protein and cell line.
|
+32 1658 9045
+32 1650 9045
