Trypsin and edta

In order to measure apoptosis, transfected CD4⁺ T cells were treated with Trypsin and EDTA (Gibco), re‐suspended in PBS (Gibco) and incubated with Annexin V‐FITC and propidium iodide (PI) from FITC Annexin V/Dead Cell Apoptosis kit (Invitrogen) according to manufacturer protocol. Counting 5x10⁴ cells at each point was performed by flow cytometry.
To measure cell cycle cells were fixed in 70% ethanol and treated with FxCycle PI/RNase Staining Solution (Thermo Scientific Inc.) according to manufacturer protocol. Cell cycle was measured by flow cytometry upon detection of propidium iodide signal.

Polycarbonate porous membrane (Whatman, UK), Polydimethylsiloxane (PDMS, Dow Corning, USA), SU-8 3035 negative photoresist (MicroChem, USA), Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA), fetal bovine serum (FBS, Gibco, USA), trypsin and EDTA (Gibco, USA), rat tail type-I collagen (BD, USA), Calcein/PI cell viability assay kit (Beyotime, China), cell counting kit-8 (CCK-8, APExBIO, USA), Annexin V-FITC apoptosis detection kit (Beyotime, China), Capecitabine (CAP, purity≥98%, Sigma, USA), Re, Rg3(S), Rh2(S) and CK ginsenosides (purity≥98%) were purchased from Sichuan China. All chemical reagents were mass spectrometry reagent grade.

The cell injections were performed on the eighth day post contusion induction. The rats were re-anesthetized as previously described[1 ], and the laminectomy sites were re-exposed again. From the cell culture plates, the BrdU-labeled cells (BMSCs and NSCs) were collected as a single-cell suspension using Trypsin and EDTA (Gibco). Using a 30-G needle and a nanoinjector pump (Stoelting Co., Wood Dale, IL, US), each cell injection was performed at the rate of 25 µl/min. Each rat from the CB, CNSCs, and COLCs groups was intraspinally injected with 300,000 cells/9 μl normal saline at the epicenter, rostral, and the caudal regions of the treatment site (each site received 100,000 cells/3 μl). In the CN group, only 9 μl normal saline was injected under the aforementioned conditions. The rats were allowed to survive 11 weeks post transplantation.