Supplementary informatiom
Collision of millimetre droplets induces DNA and protein transfection into cells
Kazuto Ikemoto1*, Ichiro Sakata2, and Takafumi Sakai2
1
Niigata Research Laboratory, Mitsubishi Gas Chemical Company, Inc., 182
Tayuuhama Shinwari, Kita-ku, Niigata, Japan
2
Graduate School of Science and Engineering, Division of Life Science, Saitama
University, 255 Shimo-ohkubo, Saitama, Japan
*Correspondence and requests for materials should be addressed to Kazuto Ikemoto
([email protected])
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Supplementary Figures
Supplementary Figure S1: Transfecting cells with proteins
a) Photograph of the fluorescence cells containing FICT-insulin.
b) Photograph of the fluorescence cells conatining FICT-IgG.
c) Efficiency of transporting into CHO cells by electrospray (3X PBS −12 kV) and no
spray
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Supplementary Figure S2: Transfection by plasmid-containing E. coli
a) Photograph of GFP-positive CHO.
b) Efficiency of transfection by electrospray (3XPBS, -12kV) to CHO and plasmid-
containing E. coli. Overnight culture with E. coli containing pEGFP-N1 was
suspended in PBS after washing twice with 0.6% calcium chloride and incubation in
methanol for one hour. Electrophoresis indicates that this suspension has a plasmid
content of 25 μg/mL, and there is no elution of plasmid from cells into the liquid.
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Electrospray of 3X PBS at −12 kV was used in the water-replaced suspension (50
μl).
c) All plasmid was present in the bacteria cells in the suspension. There was no elution
of plasmid after hypotonic treatment.
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Supplementary Figure S3: Transfection by Atomizer, free-falling and Gas flow
a) Photograph of fragrance atomizer.
b) Microphotograph of GFP expression in CHO cells sprayed by fragrance atomizer.
(PBS)
c) Transfection results (n = 3) for fragrance atomizer and free falling.
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d) Transfection results of gas flow. Air and carbon dioxide (CO2) were used by scan
device (3 mm interval, 1 cm high). DME: dimethylether and 10% Freon mixed gas
(using an air duster can).
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Supplementary Figure S4: FITC-glucosamine
Experiment 1 (Introduction of FITC-glucosamine into cells according to the present
invention)
(1) Synthesis of FITC-glucosamine
One hundred and ten (110) milligrams of glucosamine hydrochloride was dissolved in 2
mL of water, and 29 mg of fluorescein isothiocyanate (FITC) was dissolved in 4 grams
of DMSO. They were mixed together and left to stand at 30°C for 3 days. The mixture
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was heated to 90°C in an evaporator to remove the solvent, and then supplemented with
water to precipitate a yellow solid. It was then centrifuged, and the pellet was suspended
in 10 mL of water to obtain an 11 mg/mL FITC-glucosamine solution.
(2) Introduction of FITC-glucosamine into cells
The FITC-glucosamine solution obtained in (1) above was diluted with water to 0.55
mg/mL, and then 100 ml of the resulting solution was added to CHO cells. After the
mixture was left to stand for 5 minutes, a X3PBS which had a three-fold concentration
of the phosphate buffer (PBS) was sprayed at 6 mL/h under the voltage of –12 kV.
Thereafter, the cells were washed with PBS and observed under a fluorescence
microscope to count the number of fluorescent cells. The result is shown in the bar titled
“dye in dish” of Figure 1 in which the number of fluorescent cells was shown supposing
that the number in Experiment 1 was 100.
Experiment 2
Experiment was carried out as described in the Experiment 1, except that water was
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added to CHO cells instead of the FITC-glucosamine solution, and a X3PBS containing
FITC-glucosamine was sprayed instead of the X3PBS free from FITC-glucosamine.
The result is shown in the bar titled “dye spray” in the figure.
Experiment 3 (Control)
Experiment was carried out as described in the Experiment 1, except that no spraying
was carried out. The result is shown in the bar titled “control” in the figure.
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