Lipid-Assisted Microinjection: Introducing Material into the Cytosol and Membranes of Small Cells

Abstract

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

“…Previous studies of cell fusion have demonstrated that in addition to mammalian cells of other phenotypes, synthetic vesicles as well as plant protoplasts can be fused to mammalian cells (20). Synthetic vesicles have been fused with cells by using some kind of chemical treatment or such, for example PEG (21).…”

Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists

“…SLAM of loosely adherent neutrophils was achieved using lipidcoated micro-pipettes using an internal pressure of 0.5-2.0 MPa (5-20 mbar) as described previously [9]. Gentle contact between the micro-pipette and the neutrophil plasma membrane resulted in fusion of the lipid at the tip of the micro-pipette with the cell and transfer of the aqueous contents of the micro-pipette to the cytosol.…”

“…Gentle contact between the micro-pipette and the neutrophil plasma membrane resulted in fusion of the lipid at the tip of the micro-pipette with the cell and transfer of the aqueous contents of the micro-pipette to the cytosol. The pressure was held constant and the contact time between the micro-pipette and the cell was used to control the amount of injected material, as this procedure had no effect on cell viability [9]. Quantification of the amount injected was achieved by visualizing the transfer of Lucifer Yellow, which was used as an aqueous marker [9].…”

“…Agents including GTP and calmodulin [1] and an unknown diffusible factor [5][6][7][8] have also been implicated as mediators of Ca# + -channel opening. Using a new micro-injection technique [9], we have introduced heparin, albumin conjugated-heparin and anti-IP $ R antibody into the cytosol of neutrophils, a cell type that displays capacitative Ca# + entry. These strategies were aimed at inhibiting IP $ binding within the cell.…”

Electrochemical attosyringe

“…However, it is difficult to control either the location of the pipette tip during the stab injection or the amount of the injected material (5). These problems, along with a relatively large pipette tip size (usually micrometers) and high volume of the injected solution, often cause damage to cells and dramatically decrease the injection success rate (6).…”