Single-Cell Western Blotting to Determine Cell Heterogeneity

Inflammation sites contain various immune cells, including monocytes, macrophages, and leukocytes.

To identify a heterogeneous cell population by single-cell western blotting, take a pre-hydrated blotting chip, consisting of a photoactivatable polyacrylamide gel patterned into blocks. Each block has numerous microwells. Patterning into several blocks captures multiple cells in parallel.

Add a suspension containing a heterogeneous cell population. The well's dimensions allow single cells to settle. Immerse the chip into an electrophoresis chamber filled with an appropriate buffer. The detergent molecules in the buffer disrupt the cell's membrane, releasing cellular contents.

Apply an electric field. The released proteins migrate across the gel, separating into distinct-sized bands. Wash the chip with a washing buffer to remove non-specifically bound molecules, leaving only cellular proteins, including the desired marker proteins.

Expose the gel to UV light, activating its surface and immobilizing the proteins.

Add a cocktail of primary antibodies that bind specifically to marker proteins distinctive to each cell type, forming protein-primary antibody complexes. Dispense a mixture of fluorescent-tagged secondary antibodies, which bind their respective primary antibodies, labeling the corresponding cell-specific marker protein.

Place the chip in a scanner. Measure the fluorescence intensity which is proportional to the expression level of the corresponding protein marker specific for a single cell of the sample cell suspension.

For a single-cell Western blot analysis, add 1 milliliter of diluted single-cell suspension to a rehydrated single-cell Western blot chip in the bottom of a 10-centimeter Petri dish. After five to fifteen minutes, examine the chip under brightfield microscopy. Approximately 15% to 20% of the microwells should be occupied by a single cell, and fewer than 2% of the wells should contain two or more cells.

If the chip has been properly loaded, tilt the dish 45 degrees, and wash the chip three times with suspension buffer to remove any uncaptured cells. After the last wash, carefully load the chip into the electrophoresis cell of a single-cell Western instrument gel-side up, and cover the entire single-cell Western blot chip with lysis/running buffer. Initiate the cell lysis and run the instrument according to the appropriate experiment parameters.

At the end of the run, rinse the chip with two 10-minute washes with fresh washing buffer per wash at room temperature. After the second wash, add 80 microliters of the primary antibody solution of interest to the antibody-probing chamber and lower the chip gel-side down so that the antibody solution wicks across the chip.

After two hours at room temperature, wash the chip three times in washing buffer and one time in water on a shaker. Incubate the chip with an appropriate secondary antibody for one hour at room temperature, protected from light. At the end of the incubation, wash the chip three times with washing buffer, and spin the chip on a slide spinner to remove any remaining washing buffer.

To strip the single-cell Western blot chip, place a 50-milliliter tube rack in a 60-degree Celsius water bath with the water just 1 centimeter above the rack. In a fume hood, add 40 milliliters of stripping buffer, and 320 microliters of beta-mercaptoethanol to a canister. Place the chip in a 10-centimeter Petri dish inside the canister and seal the canister with parafilm.

Then, place the canister inside the tube rack in the water bath. After 90 minutes, carefully transfer the chip into a new Petri dish, and briefly wash the chip one time with wash buffer before adding 15 milliliters of fresh washing buffer to the Petri dish for a 15-minute wash on the shaker.