Bead Loading Proteins and Nucleic Acids into Adherent Human Cells

Bead loading is an inexpensive assay for loading membrane, impermeable particles into live adherent cells. This protocol has proven extremely useful for loading probes for single cell or a single molecule or fluorescence microscopy experiments. Bead loading allows researchers to quickly load all sorts of molecules, including proteins, DNA, RNA, synthetic particles, or a combination of these simultaneously into single cells.

When first performing bead loading, it is important to test out the tapping force. Cell lines respond differently to bead loading. And so the number and force of the taps can be modified for optimal cell loading while minimizing cell pealing.

To demonstrate the procedure Matthew Saxton and I will be joined by our colleagues, Dr.Amanda Cook and Gabriel Galindo. Begin by measuring approximately five milliliters of glass beads in a 50 milliliter conical tube. Add 25 milliliters of two molar sodium hydroxide to the tube and mix gently for two hours using a shaker or a rotor.

Spin down the tube of beads briefly in a centrifuge if the beads are in suspension, then decant the sodium hydroxide while retaining as many beads as possible. Wash the beads thoroughly with cell culture grade water until the pH is neutral and decant the water each time. Use the pH test strip on the effluent to confirm a neutral pH, then wash the beads thoroughly with 100%ethanol, two to three times.

Decanting the ethanol each time. Dry the beads and sprinkle them to form a thin layer inside a sterile container. Then leave the container open inside a biosafety cabinet to air-dry the beads overnight.

Gently tap or shake the container and check that the beads have a Sandy texture without clumping or flaking to ensure that the beads are completely dry. Add the beads to the apparatus and cover the entire opening of the bead holding chamber using a polypropylene mesh or equivalent material with 105 micrometer openings to allow the beads to pass through. Clamp the mesh between the male and female ends of a metal reusable imaging chamber, then seal it tightly with the waxy film and UV sterilize the apparatus for 15 minutes.

The bead loader apparatus can now be used to perform hundreds of loading assays. Store the apparatus in a dry container desiccated by silica gel or other desiccant medium and seal it. If the beads become dump thoroughly dry and sterilize the bead loader and replace it with fresh beads, as demonstrated earlier.

Remove the medium from the cells and gently aspirate all medium from around the edges of the chamber. Then tilt the chamber at approximately a 45 degree angle and remove the remaining drop of media in the center micro well. Pipette the bead loading solution gently onto the glass micro well present in the center of the chamber.

Use the bead loading apparatus to gently disperse a monolayer of glass beads on top of the cells. Ensure that the beads cover the cells completely. Pinch the chamber with two fingers, lift it around two inches and bring it down firmly using a force approximately equivalent to dropping the dish from that height gently add the medium back into the chamber by pipetting slowly onto the plastic side of the chamber.

Aspirate any floating beads without disturbing the cells, the entire bead loading procedure should be performed relatively quickly. So the cells do not dry out when they are without medium. Then incubate the cells 4.5 to two hours in the incubator.

Before imaging wash the cells three times with the medium to remove beads and excess loading components in the loading solution. Image the cells immediately or when required by the experiment using instructions in the text manuscript. Cells that were successfully bead loaded, almost always had Cy3-Pa57 and A488-H3K11ac labeled the proteins together.

One microgram of plasmid DNA and coding GFP and 0.5 micrograms of Cy3 labeled protein was also introduced into the cells via bead loading expressed and visualized. 40%of the cells were bead loaded with fab protein and 21%of the bead loaded cells express the core loaded Plasmid. The bead loaded cells expressed varying levels of plasmid.

The result of that Fisher ratio test showed that although proteins one and two had similar intensity distributions, each protein had a significantly smaller distribution than the plasmid. The levels of bead loaded proteins had little cell to cell variance and the levels of two simultaneously loaded proteins were highly correlated with each other. Proper bead loading had almost no noticeable effect on the number of human U2OS cells or their morphology when imaged directly before, directly after and 24 hours after bead loading.

Poor bead loading with excessive beads and tapping force caused cell loss, poor cell morphology and clusters of beads remaining on the cover glass. even though cells are thought to undergo mechanical damage during bead loading cells grew and proliferated in the properly bead loaded chamber. It was also observed that bead loaded cells, undergo cell division.

RPE1 and a HeLa cell lines were bead loaded with fab to demonstrate the versatility of the bead loading technique. U2OS cells were also bead loaded with a Cy5-RNA 9-mer, and Cy3-DNA 28-mer together. When attempting this protocol ensure to add only a monolayer of beads to the cells since too many beads cause cells to detach.

So after a brief 30 minute recovery post to bead loading cells are ready for imaging either immediately or hours to days later, if the procedure requires further steps such as staining, that can be done after the recovery period as well.