Thunder imager live cell 3d cell culture 3d assay

For immunostaining of whole µMOs without microsectioning, whole µMOs were fixed with 4% paraformaldehyde (Sigma) for 15–20 min at room temperature and washed three times with PBST containing DPBS supplemented with 0.1% Triton X-100. Fixed µMOs were transferred into blocking solution containing DPBS supplemented with 6% BSA (Sigma), 0.2% Triton X-100, and 0.01% sodium azide (Biosolution) and then incubated overnight on rocking platform mixer at room temperature. The samples were incubated with primary antibodies for 48 hr on a rocking platform mixer at room temperature. After washing three times with PBST, µMOs were incubated with fluorescence-labeled secondary antibody and TOPRO-3 on a rocking platform mixer for 48 hr at room temperature. After rinsing three times with PBST, 96-well plates containing µMOs were imaged using a THUNDER Imager Live Cell & 3D Cell Culture & 3D Assay (Leica Microsystems). Cell imaging and visualization of optical sections in 3D were carried out on a Leica DMi8 microscope using the Thunder Imaging System (Leica Microsystems). Stacks were taken at 2 μm intervals in the z-axis. To quantify the mean brightness of TH and MAP2 signals from each µMO, the images of individual µMO were analyzed using with Image J software (https://imagej.nih.gov/ij/download.html).

For immunostaining of whole µMOs without microsectioning, whole µMOs were fixed with 4% paraformaldehyde (Sigma) for 15–20 min at room temperature and washed three times with PBST containing DPBS supplemented with 0.1% Triton X-100. Fixed µMOs were transferred into blocking solution containing DPBS supplemented with 6% BSA (Sigma), 0.2% Triton X-100, and 0.01% sodium azide (Biosolution) and then incubated overnight on rocking platform mixer at room temperature. The samples were incubated with primary antibodies for 48 hr on a rocking platform mixer at room temperature. After washing three times with PBST, µMOs were incubated with fluorescence-labeled secondary antibody and TOPRO-3 on a rocking platform mixer for 48 hr at room temperature. After rinsing three times with PBST, 96-well plates containing µMOs were imaged using a THUNDER Imager Live Cell & 3D Cell Culture & 3D Assay (Leica Microsystems). Cell imaging and visualization of optical sections in 3D were carried out on a Leica DMi8 microscope using the Thunder Imaging System (Leica Microsystems). Stacks were taken at 2 μm intervals in the z-axis. To quantify the mean brightness of TH and MAP2 signals from each µMO, the images of individual µMO were analyzed using with Image J software (https://imagej.nih.gov/ij/download.html).