Syringe loading

Laboratory method using porous polymer adsorbent tubes, thermal desorption and gas chromatt raphy MDHS 32 Dioctyl phthalates in air Laboratory method using Tenax adsorbent tubes, solvent desorption and gas chromatography MDHS 33 Adsorbent tu standards Preparation by the syringe loading technique MDHS 34 Arsine in air Colorimetric field method using silver diethyl-dithiocarbamate in the presence of excess silver nitrate... 

MDHS33 Adsorbent tube standards (preparation by the syringe loading technique). 

A 0.5-yL syringe-loaded micro injector (Rheodyne 7520) is used for sample injection. To minimize the dead volume (< 20 nL) the fused silica column is connected directly to the injector block. 

Injection Syringe Loading Sample Injector Model 7125 (Rheodyne, USA)... 

Figure 19-25. A syringe-loading injection block. (Courtesy - Alltech Associates Inc., Deerfield, IL)...

Multiport injection valve used in HPLC to introduce the sample onto the column, the valve has six ports, two connected to a sample loop and one each to the column, pump, syringe loading port and waste. The sample is first loaded into a sample loop, usually 5-20 pi capacity, with the valve in the bypass mode rotation of the valve diverts the solvent through the sample loop whereupon the sample is swept onto the column. Sample size repeatability is better than 1%. The valves are also used for multicolumn switching. 

The 7(M0 Sanvle Irriection Vatve. Here s s 6-port sample injection valve with a removable sample loop and 7000 psi pressure rating. Size, to ii to 2.0 ml The 7120 Syringe Load Sample Injector. Fil loops conventionally or In the partial loop variable vDlume mode with only Q5 fi sample loss. 

Measure basal and Mn stimulated adenylyl cyclase activity from the same cells prepared for the cAMP mediated assay (from step 4 of Subheading 3.3.2). Transfer 600 pi of cells on ice to a 1-ml lysing syringe loaded with 600 pi of lysis buffer, and rapidly lyse the cells into a 12 x 75 mm glass tube on ice. 

Syringe Loading A Method for Inserting Macromolecules into Cells in Suspension... 

A. PREPARATION OF TISSUE CULTURED CELLS FOR SYRINGE LOADING... 

Ten-microliter volumes are syringe loaded using a MC-50 micropipette tip attached to a Gilson micropipettor. Fifty-microliter volumes are syringe loaded using a Hamilton microsyringe. 

FIGURE I Bovine aortic endothelial cells (A) and NIH fibroblast cells (B) cytoplasmically loaded with FDxLys (10 mg/ml) using the manual syringe loading technique (1-ml volume) detailed in the text. 

The method described above is a manual protocol for syringe loading of FDxLys into the cytoplasm of endothelial (Fig. lA) and fibroblast (Fig. IB) cells in vitro (Clarke and McNeil, 1992). Substitution of other macromolecules (proteins, dyes, or nucleic acids) for FDxLys will result in their loading into the cell cytoplasm by this procedure. Moreover, this technique is not limited to one or two cell types. A variety of other tissue cultured cells have been loaded in this manner, including normal rat kidney epithelial cells, QT6 cells, mouse mammary tumor cells, human angiosarcoma cells, COS-7 cells, PC 12 cells and chick neuronal cells. 

The syringe loading technique inflicts shear stress on cells to produce transient disruptions of the cell plasma membranes. As such, the amount of shear stress inflicted is of critical importance too little results in inefficient loading, too much in cell destruction. Three experimental parameters that determine the degree of shear stress inflicted are (1) the number of syringe strokes, (2) the pressure at which syringing is carried out, and (3) the internal bore size of the hypodermic needle through which the cells are passed. 

To achieve reproducible control over the amount of shear stress produced, we have built a mechanical device (Fig. 2A) that eliminates individual operator variability. Using this device, one can clearly demonstrate the relationship between loading efficiency (loading index), number of strokes, and stroke pressure (Fig. 2B). The use of this device to load FDxLys and IgG is illustrated in Fig. 3. We are presently investigating syringe loading as a cell transfection technique (Fig. 3C). 

It is very important to maintain the temperature of the cell sample as close to 37°C as possible, before, during, and after syringe loading, as cells do not survive membrane disruptions at room temperature. 

Clarke, M. S. F., and McNeil, P. L. (1992) Syringe loading introduces macromolecules into living cell cytosol. /. Cell Set. 102, 533-541. 

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