PubMed Health⌕ Search

Biomedical subjects

L Bildirici

Publications and source records attributed to L Bildirici.

5 recordsLinked to original sources
PubMed2005

Transfection of human peripheral blood mononuclear cells using immunoporation.

Immunoporation has been found to be able to efficiently transfect a wide range of human cultured cell lines. This report shows that peripheral blood mononuclear cells can also be efficiently transfected using immunoporation. The immunoporation of the cells with fluorescent TMR-Dextran, using Immunofect MG beads, indicates that transient holes of 5.4nm in diameter or larger are formed during immunoporation. The efficiencies of transfection of lymphocytes transfected with vectors coding for EGFP and lacZ were found to be within the range of 15-30% with high levels of cell viability of more than 90%. In addition, it was observed that mononuclear cells stimulated with PHA expressed transfected reporter genes with a higher efficiency. In conclusion, these results demonstrate that immunoporation using Immunofect MG beads can be used for the efficient transfection of primary lymphocytes with DNA or other macromolecules.

Antibodies↗
PubMed2004

Factors that affect the efficiency of cell transfection by immunoporation.

Immunoporation is a recently discovered method that is able to transfect various human cell lines efficiently by targeting the cell surface antigens with antibody-coated beads. For this particular study, HL60, a cell line difficult to transfect by other methods, was used as a model to define the various parameters of the cell membrane that determine the efficiency of this method. The level of antigen expression on the cell surface was the first parameter to be analyzed and experiments indicated that there is a close correlation between the level of expression of surface antigens and the efficiency of immunoporation. The mixing speed, the bead to cell ratio, and the mixing time were all found to affect the ability of the antigen-coated beads to pull holes in the cells and it was found that for HL60 cells the optimum mixing speed was 40 rpm and the bead to cell ratio was 20:1 using a mixing time of 6 h.

Antigen-Antibody Reactions↗
PubMed2001

An investigation into the suitability of silica beads for cell separations based on density perturbation.

This study has investigated possible alternative types of beads for fractionating cells on the basis of density perturbation. It is well known that uniform magnetic beads can be extremely important tools for separating cells by both magnetic separation techniques and density perturbation. However, because of the inherent expense associated with the use of magnetic beads, it was decided to study the possible use of inexpensive silica beads for density perturbation in terms of their attachment and modification of density of cells and to compare them with uniform Dynabeads. Silica beads were analyzed to determine their size and effect on the density of cells. Differentiated HL60 cells were used as a model system. As differentiation occurs, different levels of antigens are expressed on the cell surface and this results in different numbers of beads binding to cells. DMSO-differentiated HL60 cells were mixed with anti-CD11b-coated beads at a ratio of 20:1 (beads/cell), and gentle mixing was carried out at 20 degrees C on the end-over-end mixer. The binding of antibody-coated silica beads and Dynabeads to partially differentiated HL60 cells were compared. The conclusions reached on the basis of these experiments are that antibody-coated silica beads (Ab-coated silica) can be used as alternative beads for some cell fractionations. However, compared with Dynabeads, there are more beads that are only transiently associated with cells, possibly indicating that higher levels of detachment of beads from cells occur when silica beads are used. In addition, silica beads are usually heterogeneous in size and this would make it difficult to use these beads for the isolation of purified subpopulations of differentiated cells.

Cell Count↗
PubMed2000

Fractionation of differentiating cells using density perturbation.

This paper describes the development of a new method for the fractionation of purified subpopulations of partially differentiated cells on continuous isopycnic gradients, using a density perturbation method based on the ability of cells to bind dense antibody-coated beads. Until now none of the available fractionation techniques, such as magnetic cell fractionation has been efficient for separating subpopulations of partially differentiated cells. The fractionation experiments described in this report used promyelocytic HL-60 and DMSO-induced granulocytic HL-60 cells as a model system. Populations of cells, modified by the binding of dense beads were fractionated on isotonic, isopycnic Optiprep gradients by centrifugation at 220xg for 90 min at 20 degrees C. Examination of the different gradient fractions showed that, as cells bind increasing numbers of beads, they are found in the denser regions of the isopycnic gradients. Indirect immunofluorescence was combined with flow cytometric techniques to characterise the fractionation of partially differentiated cells. Flow cytometric results confirmed that as antigenic determinants appear on the surface at higher levels of expression, the number of beads binding to each cell increased. Furthermore, after fractionation, when the bead-bound and non-bead-bound cells were cultured in the presence of DMSO, those cells that had bound more beads targeted to differentiated cells were found to achieve terminal differentiation faster than those cells that had not been associated with any beads.

Cell Differentiation↗