Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

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

The production of an amine-modified derivative of 5-aminosalicylic acid by activated neutrophils Roles for myeloperoxidase and chloride ions

Following incubation with activated neutrophils, two metabolites of 5-aminosalicylic acid (5-ASA) were identified by HPLC. These two metabolites accounted for approximately 60% and 20% of the original 5-ASA. The formation of the major metabolite was prevented by pre-incubation with the peroxidase inhibitor, azide, and reduced by the omission of chloride ions from the incubation medium, or the presence of catalase. A similar product was generated by sodium hypochlorite or myeloperoxidase/H2O2, mass spectroscopical analysis being consistent with it being 5-nitroso-salicylate. Our finding suggests that the efficacy of 5-ASA results from its ability to react with and so scavenge hypochlorite ions. The amount of amine-modified 5-ASA in the faecal stream may thus provide an indicator for hypochlorite production in the bowel

Exclusion of exogenous phosphatidylinositol‐3,4,5‐trisphosphate from neutrophil‐polarizing pseudopodia: stabilization of the uropod and cell polarity

Although there is accumulating evidence that the generation and localization of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have important functions in neutrophil polarization and chemotaxis, the mechanism of this linkage has yet to be established. Here, using exogenous fluorescent PtdIns(3,4,5)P(3) introduced into the inner leaflet of the neutrophil plasma membrane by a cationic carrier, we show that: first, PtdIns(3,4,5)P(3) uniformly delivered to the neutrophil plasma membrane is excluded from newly forming pseudopodia; second, PtdIns(3,4,5)P(3) translocates to and is immobilized at the pole opposite a stable polarizing pseudopod; third, asymmetric delivery of PtdIns(3,4,5)P(3) to the neutrophil triggers the generation of polarizing pseudopodia at the opposite pole; and finally, PtdIns(3,4,5)P(3) triggers repetitive Ca(2+) signals, the onset of which precedes morphological polarization. These data suggest that translocation and immobilization of PtdIns(3,4,5)P(3) or a 3,x-phosphorylated metabolite in the uropod functions as an important polarization cue that defines neutrophil polarity and stabilizes the generation of pseudopodia at the opposite pole

Microinjection and micropipette-controlled phagocytosis methods for neutrophils

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here we outline two techniques which work well with neutrophils, namely, SLAM (Simple Lipid-Assisted Microinjection) and electromicroinjection. As these methods utilize micropipettes, we also include a simple method which uses a micropipette to deliver a phagocytic stimulus to a specific cell at a defined time, enable detailed study of the phagocytic process from particle contact to particle internalization

Optical methods for the measurement and manipulation of cytosolic calcium signals in neutrophils

The measurement and manipulation of cytosolic free Ca2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca2+ signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils, and are discussed here, especially the use of fluorescent indicators of Ca2+ and photoactivation of reagents involved in Ca2+ signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we outline some of the techniques that have been used to monitor, visualize, and manipulate Ca2+ in neutrophils

CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4·4 mm MgCl(2)/20 mm EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 ± 13% and 71 ± 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 ± 5%) and C9 (95 ± 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation