SugarFect®

SFE0911-1

For reserch use only

Gene Transfection Reagent for Stem cells & Primary Cells

SF-P50 ………

SugarFect P50(1.0mL)

INTRODUCTION

1. Dilute SugarFect reagent to desired concentration (refer to theSugarFect is a non-viral vector made of cationized polysaccharide

table 1 “N/P Ratio” in the Appendix section) using sterilized

for gene transfection of eukaryotic cells. This reagent uses sugarDDW.

receptor-mediated endocytosis to deliver a desired nucleic acid

2. P repa re Suga rFect-DNA complexes by addi ng diluted(DNA) into a target cell. SugarFect has shown low cytotoxicity andSugarFect reagent drop-by- drop to pDNA at 1:1 ratio (v/v) in asome good results in primacy cells and stem cells.

sterile tube.

For users requires extremely low cytotoxicity, please try ourI M PORTA N T: A lways a dd d ilut ed Suga rFect reagent

original “Subfection” method. Subfection is a one of the reverse-

drop-by-drop to diluted pDNA. Reversing the order of additiontransfection method which uses cell adhesion protein and original

could reduce the transfection efficiency.

coating solution for cell culture plate (see 2. Subfection Protocol).

3. Mix gently by tapping the tube containing the complexes.
Incubate at room temperature without agitation for the minimum

PROCEDURE
The following protocol is written for transient transfection ofplasmid DNA into adherent eukaryotic cells.

of 15 minutes (up to 1 hour) to allow formation of complexes.
► Appropriate amount of SugarFect-DNA Complexes is 50μL
per well in a 12-well plate.

► Mark denotes procedu res wr it ten for t ransfection of

4. Change cell culture medium to serum-free medium whileadherent eukaryotic cells in a 12-well plate.

waiting for SugarFect-DNA complexes to form.
►Wash cells once with 500μL of DPBS(-) and add 500 μL ofAdditional Materials Required, but Not Supplied

serum-free medium per well, return the plate to 37 oC incubator• Sterilized double distilled water (DDW)

if SugarFect-DNA complexes need more incubation time.

• Dulbecco’s Phosphate Buffered Saline (-) (DPBS)

5. Add appropriate amount of SugarFect-DNA complexes to cells• Plasmid DNA (pDNA) (100 μg/ml) dissolved in DPBS (*1)

in Serum-free medium. Incubate cells with complexes for 3-6• Sterile microcentrifuge tubes

hours under normal culture condition.

• 12-well culture plate• Culture medium►Add 50 μL of SugarFect-DNA complexes to each well.
6. Change medium back to normal culture medium.

• (For 1. Standard protocol)
Serum & antibiotics-free culture medium• (For 2. Subfection® protocol)
Cell adhesion protein (1mg/ml) (pronectin or fibronectin)►Carefully aspirate serum-free medium and wash with 500μLof DPBS(-) twice. Add 1mL of appropriate culture medium.
7. Incubate the cells at normal culture condition until the assay forgene expression is to be performed.

(*1) TE buffer is not recommended.
2. Subfection Protocol
1. Standard Protocol

Transfection can be performed in the presence of serum.

One Day before Transfection

The Day of Transfection

1. Cou nt a nd plat e enoug h cel ls to a ch ieve roug h ly 80 %1. Prepare coating solution containing cell adhesion protein in

confluency at the time of transfection. Incubate cells at normal

sterile tube.

culture condition (e.g. 37oC in a CO2 incubator) for 24 hours.
► Plate 1.25 x 10 cells in 1mL of culture medium per well in a
512-well plate.

►Dilute 20μL of Reagent A, 40μL of Cell adhesion protein(1mg/ml) to 400μL using DPBS(-).
2. Coat cell culture plate with coating solution, and incubate at
37oC for 1.5hours to allow them to absorb. Use enough volume ofThe Day of Transfection

coating solution to prevent from drying.

► Add 400μL of coating solution to each well.

► Plate 5.0 x 10 4cells in 1ml of culture medium per well in a

3. Prepare SugarFect-DNA complexes by adding diluted SugarFect
reagent drop-by- drop to pDNA at 1:1 ratio (v/v) in a sterile tube.
I M PORTA N T: A lways a dd d i lut e d Suga rFe c t r e age nt

12-well plate
7. Incubate the cells at normal culture condition until the assay forgene expression is to be performed.

REFERENCES

drop-by-drop to diluted pDNA. Reversing the order of addition
Review

could reduce the transfection efficiency.
4. Mix gently by tapping the tube containing the complexes.
Incubate at room temperature without agitation for the minimum of
15 minutes (up to 1 hour) to allow formation of complexes.
► Appropriate amount of SugarFect-DNA Complexes is 50μL

Jo J, Tabata Y.
Non-viral gene transfection technologies for genetic engineering of stem cells.
Eur J Pharm Biopharm. 2008 68(1):90-104.

Original Paper
Okazaki A, et al. A reverse transfection technology to genetically engineer
adult stem cells. Tissue Eng. 2007 13(2):245-51.

per well in a 12-well plate.
5. Coat cell culture plate with SugarFect-DNA Complexes, andincubate at 37o C for 30 minutes to allow them to absorb. Useenough volume of coating solution to prevent from drying.

Jo J, et al. Expression profile of plasmid DNA by spermine derivatives ofpullulan with different extents of spermine introduced. J Control Release.
2007 118(3):389-98.

► Dilute 50μL of SugarFect-DNA complexes to 400μL using
350μL of DPBS(-). Carefully aspirate the coating solution ofcell culture plate and wash with 500μL of DPBS(-) once. Add
400μL of SugarFect-DNA complexes solution to each well.

Okasora T, et al. Augmented anti-tumor therapy through natural targetabilityof macrophages genetically engineered by NK4 plasmid DNA. Gene Ther.
2008 15(7):524-30.

6. Count and plate cells onto the surface of substrate which was

Nagane K, et at. Practical induction system for dopamine-producing cells

coated with the SugarFect-DNA complex.

from bone marrow stromal cells using spermine-pullulan-mediated reverse
transfection method. Tissue Eng Part A. 2009 15(7):1655-65.

APPENDIX
Dulbecco’s Phosphate Buffered Saline (-) (1L)
(Sold as Dulbecco’s PBS (-)"Nissui" in Japan from Nissui Pharmaceutical Co., Ltd.)
Sodium Chloride…………………..……….. 8,000 mg
Potassium Chloride ………………………….. 200 mg
Disodium Phosphate (anhydrous)…………... 1,150 mg
Monopotassium Phosphate (anhydrous)…..…. 200 mg
Dissolve above ingredients in 1L (pH=7.35 - 7.55, osmotic pressure: 280±5mOs/kg) of double distilled water. Sterilize by autoclaving at 121
oC for 15 minutes, or by filtration.
Dilution of SugarFect (Table 1)
N/P Ratio (*)
SugarFect
DDW
Total Vol.
110 μL90 μL
100 μL220 μL80 μL
100 μL
330 μL
70 μL
100 μL* N/P (=[Amino group of SugarFect] / [Phosphate of DNA])* RatioFor stem cells, recommended N/P ratio is 3
Recommended coating solution and SugarFect-DNA complex volume (Table 2)
Coating solution for SubfectionCell Culture
Approx.
Cell adhesion
PlatesGrowth Area
ReagentA
PBS(-)
protein (1mg/ml)
296 well
0.3 cm /well
5 μL
10 μL
105 μL24 well
1.9 cm2/well
10 μL
20 μL
170 μL
12 well
3.8 cm2/well
20 μL
40 μL
340 μL
6 well9.5 cm2/well
40 μL
80 μL
680 μL

DilutedSugarFect
5 μL
12.5 μL
25 μL
50 μL

SugarFect-DNA complex
pDNA
Total
(100 μg/mL)(/well)5 μL
10 μL
12.5 μL
25 μL
25 μL
50 μL
50 μL
100 μL

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