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DNA modification of live cell surface.

Borisenko GG, Zaitseva MA, Chuvilin AN, Pozmogova GE - Nucleic Acids Res. (2009)

Bottom Line: By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface.All procedures can be completed within minutes and do not alter cell viability.Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Physical-Chemical Medicine, Russian Academy of Science, Moscow 119312, Russia. grigoryb@yahoo.com

ABSTRACT
We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell-cell interactions.

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Hybridization of T18NSte integrated into PM with complementary A25F at the cell surface. Jurkat cells were labeled with T18NSte (0.4–2.4 μM) at 37°C for 5 min, washed and incubated with complementary A25F (0.4 μM) at 37°C for 5 min. Then cells were placed in water bath to cool down to 6°C during 20 min time period. (A) Histograms of cell distribution by A25F fluorescence pretreated with T18NSte at the concentration of 0.0, 0.4 and 1.6 μM (red, black and green lines, respectively). (B) Dose dependence of a mean A25F fluorescence intensity detected from cells on T18NSte concentration (n = 3). (C–E) Microphotographs of A25F fluorescence in Jurkat cells pre-incubated with 1.6 μM T18NSte (C) and without T18NSte (D, E). (C, D) Instrumental conditions are similar; (E)—same as (D), but PMT gain is enhanced to demonstrate the presence of unstained cells (dark spots) on the fluorescent background of A25F.
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Figure 7: Hybridization of T18NSte integrated into PM with complementary A25F at the cell surface. Jurkat cells were labeled with T18NSte (0.4–2.4 μM) at 37°C for 5 min, washed and incubated with complementary A25F (0.4 μM) at 37°C for 5 min. Then cells were placed in water bath to cool down to 6°C during 20 min time period. (A) Histograms of cell distribution by A25F fluorescence pretreated with T18NSte at the concentration of 0.0, 0.4 and 1.6 μM (red, black and green lines, respectively). (B) Dose dependence of a mean A25F fluorescence intensity detected from cells on T18NSte concentration (n = 3). (C–E) Microphotographs of A25F fluorescence in Jurkat cells pre-incubated with 1.6 μM T18NSte (C) and without T18NSte (D, E). (C, D) Instrumental conditions are similar; (E)—same as (D), but PMT gain is enhanced to demonstrate the presence of unstained cells (dark spots) on the fluorescent background of A25F.

Mentions: To carry out a hybridization reaction of complementary oligonucleotides at the cell surface, we incorporated non-fluorescent T18NSte into cell PM and then co-incubated these cells with a complementary FAM-labeled oligonucleotide A25F. Fluorescence microscopy analysis revealed that only T18NSte modified cells became fluorescent (Figure 7C and D). Flow cytometry further confirmed that the entire population of cells pre-labeled with T18NSte shifts to higher fluorescence signal after incubation with A25F as compared with non-labeled cells (Figure 7A). Next, we performed hybridization reaction using constant concentration A25F and cells with various amounts of incorporated T18NSte. Fluorescence signal detected from cells depended on the concentration of T18NSte used for the labeling, and hence, it depended on the surface density of T18NSte (Figure 7B). Moreover, this reaction nearly reached the saturation when T18NSte concentration (0.8 µM) was twice A25F (0.4 µM). Stoichiometry of complimentary oligonucleotide reaction is 1:1, hence, it is possible that at least 50% of T18NSte was incorporated into PM and was available for the hybridization.Figure 7.


DNA modification of live cell surface.

Borisenko GG, Zaitseva MA, Chuvilin AN, Pozmogova GE - Nucleic Acids Res. (2009)

Hybridization of T18NSte integrated into PM with complementary A25F at the cell surface. Jurkat cells were labeled with T18NSte (0.4–2.4 μM) at 37°C for 5 min, washed and incubated with complementary A25F (0.4 μM) at 37°C for 5 min. Then cells were placed in water bath to cool down to 6°C during 20 min time period. (A) Histograms of cell distribution by A25F fluorescence pretreated with T18NSte at the concentration of 0.0, 0.4 and 1.6 μM (red, black and green lines, respectively). (B) Dose dependence of a mean A25F fluorescence intensity detected from cells on T18NSte concentration (n = 3). (C–E) Microphotographs of A25F fluorescence in Jurkat cells pre-incubated with 1.6 μM T18NSte (C) and without T18NSte (D, E). (C, D) Instrumental conditions are similar; (E)—same as (D), but PMT gain is enhanced to demonstrate the presence of unstained cells (dark spots) on the fluorescent background of A25F.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 7: Hybridization of T18NSte integrated into PM with complementary A25F at the cell surface. Jurkat cells were labeled with T18NSte (0.4–2.4 μM) at 37°C for 5 min, washed and incubated with complementary A25F (0.4 μM) at 37°C for 5 min. Then cells were placed in water bath to cool down to 6°C during 20 min time period. (A) Histograms of cell distribution by A25F fluorescence pretreated with T18NSte at the concentration of 0.0, 0.4 and 1.6 μM (red, black and green lines, respectively). (B) Dose dependence of a mean A25F fluorescence intensity detected from cells on T18NSte concentration (n = 3). (C–E) Microphotographs of A25F fluorescence in Jurkat cells pre-incubated with 1.6 μM T18NSte (C) and without T18NSte (D, E). (C, D) Instrumental conditions are similar; (E)—same as (D), but PMT gain is enhanced to demonstrate the presence of unstained cells (dark spots) on the fluorescent background of A25F.
Mentions: To carry out a hybridization reaction of complementary oligonucleotides at the cell surface, we incorporated non-fluorescent T18NSte into cell PM and then co-incubated these cells with a complementary FAM-labeled oligonucleotide A25F. Fluorescence microscopy analysis revealed that only T18NSte modified cells became fluorescent (Figure 7C and D). Flow cytometry further confirmed that the entire population of cells pre-labeled with T18NSte shifts to higher fluorescence signal after incubation with A25F as compared with non-labeled cells (Figure 7A). Next, we performed hybridization reaction using constant concentration A25F and cells with various amounts of incorporated T18NSte. Fluorescence signal detected from cells depended on the concentration of T18NSte used for the labeling, and hence, it depended on the surface density of T18NSte (Figure 7B). Moreover, this reaction nearly reached the saturation when T18NSte concentration (0.8 µM) was twice A25F (0.4 µM). Stoichiometry of complimentary oligonucleotide reaction is 1:1, hence, it is possible that at least 50% of T18NSte was incorporated into PM and was available for the hybridization.Figure 7.

Bottom Line: By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface.All procedures can be completed within minutes and do not alter cell viability.Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Physical-Chemical Medicine, Russian Academy of Science, Moscow 119312, Russia. grigoryb@yahoo.com

ABSTRACT
We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell-cell interactions.

Show MeSH
Related in: MedlinePlus