Single-Cell Western Blotting to Determine Cell Heterogeneity
Transcrição
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.
