Paul Held, Ph.D., Bridget Bishop and Peter Banks, Ph.D., BioTek Instruments
Fluorescence microscopy is a mainstay of life science and clinical research. With it, scientists can study cellular morphology, identify subcellular compartments, track specific proteins and monitor the impact of stimuli and drugs.
Traditionally fluorescence microscopy is conducted on glass slides. This method isn’t conducive to high-throughput studies and especially not to the high-content screening approaches increasingly being used in drug-discovery and biotech firms. In such applications, microplates reign supreme, and any application that can be adapted onto one benefits in terms of both ease of use and organization.
Immunofluorescence is no exception.
At BioTek Instruments, we have considerable experience automating routine laboratory processes. Here, we outline how to automate sample preparation for fluorescence microscopy.
Immunofluorescence basics
The process of fluorescence microscopy is relatively straightforward. Whether grown on a glass slide or a clear-bottomed microtiter plate, cells generally must be fixed, permeabilized with detergent and finally stained.
If your cells express a fluorescent protein, you can skip the staining step, unless you wish to identify cell structure or specific proteins, or count all cells in a population regardless of fluorescent protein expression.
To stain nuclei, you can use DAPI (4’,6-diamidino-2-phenylindole), a dye that binds nuclear DNA and which theoretically stains both live and dead cells without permeabilization. However, we prefer to stain fixed and permeabilized cells, as DAPI is less efficient at crossing live-cell membranes.
To stain cytoskeletal structures, you can use fluorescently tagged phalloidin, a peptide toxin that binds polymerized actin. And of course you can add antibodies to specific proteins of interest to identify, for instance, their subcellular localization.
After you’re finished, just view the microplate under a microscope, excite at the appropriate wavelengths, take a picture and you’re done.
Fixation and permeabilization
If your automation system uses both peristaltic-pump and syringe-pump dispensers, as BioTek’s EL406 Combination Washer Dispenser or MultiFlo FX Microplate Dispenser does, use the peri pump for precious reagents, such as antibodies, and the syringe pump for less expensive materials. (Peristaltic pumps can be reversed to recover unused reagents.)
Use the syringe pump to dispense fixation solution. We suggest 100 μL of 4% paraformaldehyde for 10 minutes at room temperature, followed by two 200-μL washes in buffered saline. Then permeabilize, also using the syringe pump, with 50 μL of buffered saline containing 0.1% Triton X-100 for 10 minutes.
Staining
After you’ve completed the fixation and permeabilization steps, you’re ready to stain. Use a peristaltic pump, if available.
Say you want to stain cells for nuclei with DAPI, the actin using phalloidin, and the mitochondria using antibodies. Do the antibody staining first, applying 50 μL of diluted primary antibody (try 1:750) for one hour at room temperature. Wash once with buffered saline containing Triton X-100, then apply your labeled secondary antibody (50 μL, 1:500 dilution). Incubate 60 minutes at room temperature and wash twice with buffer plus detergent. Then add phalloidin (50 μL, 10 minutes at room temperature), wash and add DAPI (50 μL, 10 minutes at room temperature). Wash once more with buffered saline and add 100 uL buffered saline to keep the cells hydrated. You’re ready to image.
Of course, each automation platform, cell system and staining reagent varies, so be sure to tailor your particular conditions to the experimental conditions.
Automation considerations
Automation offers several advantages over manual staining. Most obviously, it’s a time saver, freeing you to take on other tasks while the instrument performs previously manual tasks.
Throughput is another advantage, as most instruments can handle cells in either 96- or 384-well plates. Because they frequently also feature multichannel heads for washing and dispensing, automated liquid handlers generally are faster than manual pipetting, as well as more accurate and reproducible. Experimental organization is yet another advantage. Microplates make organization and keeping track of samples and the resultant data easy.
On the other hand, cells are fragile and care must be taken in programming an automated liquid handler to avoid dislodging or harming them.
One solution is to leave a small amount of residual fluid in the wells after aspirating off the fluid, to minimize the impact of adding new reagent. You also can tinker with the system’s run program to modify the horizontal position of the dispensing and wash heads to be closer to the walls of the well than to its center and to adjust the rates of dispensing and aspiration.
It might take a little optimization to get the process working perfectly, though your automation vendor’s application support specialists should be able to help. But once you get the process running right, you need never stain your cells manually again.
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