Quantitative microinjection using fluorescence calibration of streaming microdroplets on a superhydrophobic surface

• Published in: Experimental cell research 2018-09, Vol.370 (2), p.426-433
• Main Authors: Moore, Christopher L., Taylor, Erin M., Ball, Kelly K., Bernock, Laura J., Griffin, Robert J., Jung, Seunghyun, Shoeib, Amal, Borrelli, Michael J.
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; CARCINOMAS; Cells; DEXTRAN; FLUORESCENCE; Microdroplets; Microinjection; OILS; PROSTATE; Quantitative; Superhydrophobic
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2018.07.006

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. [Display omitted] •Superhydrophobic surfaces prevent droplet surface adhesion and deformation.•Wide tipped micropipettes create a stream of microdroplets.•Streaming creates a large number of varying sized droplets quickly and easily.•Fluorescence-volume calibration curves can be created with tracer microdroplets.•Calibrated fluorescent tracers allow for quantifiable, cellular microinjection.