Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the
absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate
during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate
two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic
imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The
proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to
support protoplasts in the form of uniform thin layer, which has three unique properties.