Home Up Feedback Contents Search Sell your Prod. Meet us News           

 
Home • Up

Article 2

 

Home

GENTAUR

+32 1658 9045

or

0032 (0)16 41 44 07

+32 1650 9045

info@gentaur.com

Av. de l' Arme 68

B-1040 Brussels

BELGIUM

France

tel 01 43 25 01 50

fax01 43 25 01 60

9, rue Lagrange

75005 Paris

Italia

tel 02 36 00 65 93

fax 02 36 00 65 94

20135 Milano

Deutschland

tel +32 1658 9045

fax +32 1650 9045

Polska

Tel 058 710 33 44

Fax 00 32 16 50 90 45

ul. Grunwaldzka 88A/2

81-771 Sopot

日本

tel +81 78 386 0860

fax +81 78 306 0296

Minaatojimaminami-manchi

Chuo-ku, Kobe

065-0047

sterreich
+43720880899

Canada Montreal
+15149077481

Česk republika Praha
+420246019719

Danmark
+4569918806

Finland Helsset
+358942419041

Ελλάς Αθήνα
+302111768494

Magyarorszg Budapest
+3619980547

Ireland Dublin
+35316526556

Luxembourg
+35220880274

Nederland
+31208080893

Norge Oslo
+4721031366

Polska Warszawa
+48223988221

Sverige Stockholm
+46852503438

Schweiz Zri
+41435006251

US New York
+17185132983

Other Countries
0032 (0)16 41 44 07


 

 

 

 

 

Clonagen

Gentaur

Genprice

Bioxys

Labprice

Delivery of IgG-FITC using SAINT-PhD  
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 10L SAINT-PhD reagent, the protein of interest, and HBS buffer as diluent in
a total volume of 25L/well. Protein complex formation occurred immediately and an additional
100L 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 (125L) was added to each well. The cells were incubated with the mixture for 4 hours
(37C, 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 (37C, 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 10L of SAINT-PhD reagent in a total volume of 25L. The complexes were filled
up to 125L with total growth medium and added to the cells in a 48-well plate. The complexes were
incubated for 4 hours (37C,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 25L antibody/HBS/SAINT-PhD complex drop wise to each well with cells and incubated
at 37C 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 10L of SAINT-PhD reagent in a total volume of 25L. The complexes were added
directly to the cells in a 48-well plate. The complexes were incubated for 4 hours (37C,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 350L per well. So, with the complex added, the end-volume is 375L.
In the standard delivery procedure the end-volume is 125L, 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 10L of SAINT-PhD reagent in a total volume of 25L. The complexes were added directly
to the cells in a 48-well plate. The complexes were incubated for 24 hours (37C,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.

 

 

Send mail to webmaster@gentaur.com with questions or comments about this web site.
Copyright 2002 GENTAUR Molecular Products
Last modified: 05/29/09