cell type: Schwann cells time point: 14d tissue: Nerve
Treatment protocol
In the co-cultures, appropriate SCs (Myelinating SCs at different stages) were selected on a computer-controlled inverted microscope with a stage incubator that creates a physiological atmosphere for the preparations (Cell Observer, Zeiss, Oberkochen, Germany); their coordinates were recorded to serve as a reference. During selection, any SCs exhibiting signs of stress, such as vacuoles, blebs, deformed membranes, were excluded. For laser capture microdissection (LCM), we used a Leica laser microdissection systems (LMD7000, Leica). The myelinating SCs at different stages were dissected by UV laser (laser power set to 2.2-3.2) and attached to CapSure LCM caps with help of infrared laser (power set to 80%). Caps were collected in 0.5 ml eppendorf tubes containing 100μl of RNAlater buffer (QIAGEN) and stored upside down at -80°C until RNA isolation.
Growth protocol
Rat Schwann cells and dorsal root ganglion (DRG) neurons were isolated and cocultured in DMEM-HG medium with 10% FBS, and allowed to attach overnight. The next day, the media was replaced with DRG growth medium for 2 d to allow the Schwann cells to repopulate the neurites and then switched to the DMEM medium supplemented with 50 ng/ml NGF, ITS (Sigma), and 0.2% bovine serum albumin (BSA) (Sigma) for about 4 d to initiate basal lamina formation, then the premyelinated cocultures were maintained in DMEM medium containing 15% FBS, 50 ng/ml NGF and 50 μg/ml L-ascorbic acid (AA) (Sigma) for 2-3 w.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US)following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany)and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit.
Description
Gene expression in Schwann cells after co-cultured with DRG neurons for14 days
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, GeneSpring Software 12.6.1 (Agilent technologies, Santa Clara, CA, US
