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A simple and reproducible procedure was developed to measure the volume of liquid microinjected into cells. A calibration curve of droplet fluorescence intensity versus volume was constructed by injecting a fluorescent dextran solution through a 125–150 µm diameter micropipette into an oil-filled culture dish to create a spray of varied-sized droplets. The droplets retained a spherical shape because they were in an oil medium and they settled onto a glass surface coated with a superhydrophobic surface. Fluorescent micrographs of the droplets were obtained and analyzed with Image-J software to quantify the fluorescence intensity and radius of each spherical droplet to produce the calibration curve. Subsequently, Dut-145 human prostate carcinoma cells were microinjected with the same fluorescent dextran solution and fluorescent micrographs of the cells were obtained using the identical exposure conditions used to photograph the droplets. The measured fluorescence intensity of the microinjected cells was entered into the formula for the regression line that was fit to the calibration curve allowing determination of the volume of solution injected into each cell. Thus, a mixture consisting of known concentrations of a test material of test material (macromolecules, drugs, etc.) and a fluorescent dextran, volumetric, tracer can be used to quantify the relationship between the amount of a microinjected material and subsequent effects on cells.

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.

A modification of the protocol developed by Kawamoto, J C & Barrett, J N, Brain res (1986) , in press [3] for freezing primary neuron cultures in a solution containing low sodium and high lactate and potassium concentrations was used to freeze synchronous mitotic and G1 CHO cells. After thawing, the cells behaved as if they had never been frozen with respect to cell growth, cell division, plating efficiency, and hyperthermic sensitivity.