Immunocytochemistry and fluorescence microscopy

To compare undifferentiated and differentiated cells, it is important to evaluate the phenotypic properties of mNPCs under pulsed DC stimulation. The phenotypic markers used in our study included nestin, Sox2, Tuj-1, GFAP, and O4. Nestin is expressed in early neural lineage and differentiated insulin-secreting cells [32,33]. Expression of the transcription factor Sox2 is commonly used as a neural plate or early neural marker and is essential for neurogenesis in the CNS [34]. The immature neuronal Tuj-1 is expressed almost exclusively in early neuronal differentiation [35]. GFAP is expressed in neural tissues and distinguishes astrocytes from other glial cells during CNS development [36]. The oligodendrocyte marker O4 is an antigen on the surface of oligodendrocyte progenitors. O4 has commonly been used as the earliest recognized marker specific for the oligodendroglial lineage [37].

After 3, 7, or 14 DIV culturing after seeding, the cells were rinsed with PBS and fixed with 4% paraformaldehyde (PFA). PBS and PFA were pumped into the chip at a flow rate of 25 μl/min for 20 min. Cells were then permeabilized with 0.1% Triton X-100. Triton X-100 was pumped into the chip at a flow rate of 50 μl/min for 6 min to replace the PBS and PFA. Next, the Triton X-100 was pumped into the chip slowly at a flow rate of 50 μl/h for an additional 30 min to react with the cells. The cells were then blocked with PBS containing 1% bovine serum albumin (BSA) to reduce nonspecific antibody binding. The BSA was pumped at a flow rate of 50 μl/min for 6 min to replace the Triton X-100. The flow rate was next reduced to 100 μl/h and then pumped for 1 h. The cells were subsequently detected by double immunostaining using the following three combinations: (1) nestin (to identify early neural cells) (mouse anti-nestin, 1:1000; abcam, Cambridge, MA) and Sox2 (to identify early neural cells) (rabbit anti-Sox2, 1:1000; abcam), (2) neuron-specific class III beta-tubulin (Tuj1) (to identify neurons) (rabbit anti-Tuj1, 1:500; abcam) and glial fibrillary acidic protein (GFAP) (to identify astrocytes) (mouse anti-GFAP, 1:1000; eBioscience, San Diego, CA), or (3) Tuj1 (to identify neurons) (rabbit anti-Tuj1, 1:500; abcam) and O4 (to identify oligodendrocytes) (mouse anti-O4, 1:500; R&D Systems, Minneapolis, MN). The antibodies for double immunostaining were first pumped into the chip, and then the chip was incubated for 18 h at 4°C. The reagents used for immunostaining were all diluted with PBS. After a washing step with PBS, Alexa Fluor–conjugated secondary antibodies (1:800; Thermo Fisher Scientific) were applied to the cells for 1 h at room temperature (RT) in the dark. Cells were then rinsed with PBS. PBS was pumped at a flow rate of 50 μl/min for 15 min. For nuclear staining, Hoechst 33342 (1:1000) was pumped into the chip and incubated for 10 min at RT. The cells were then washed by pumping PBS into the chip at 50 μl/min for 10 min. After immunostaining, the cells were examined using a confocal fluorescence microscope (TCS SP5).