Mtt kit

All chemicals needed for the synthesis of IPP-c nanoparticles (NPs) were commercially purchased and could be used without any refinement unless otherwise noted. Dopamine-hydrochloride (DA-HCl), K₂PtCl₄ and Indocyanine green (ICG) were obtained from Sigma-Aldrich (Shanghai, China). The sulphydryl polyethylene glycol (PEG-SH, 5 KDa) was purchased from Aladdin (Shanghai, China). The anti-CXCR4 (ab208128) was bought from Abcam (Shanghai, China). The cell culture medium, PBS, trypsin and MTT kit were purchased from Thermo Fisher Scientific (Hong Kong, China). Matrigel^® Matrix was bought from Becton Dickinson (America). D-Luciferin was obtained from Gold Biotechnology (America). The synthesis process of IPP-c NPs was shown in the Supporting Information.

Cell viability and proliferation assays were done as previously described.
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Briefly, cells were seeded at 1 × 10
⁴cells per well in 96-well plates and cultured for 72 hours prior to testing using an MTT kit (Thermo Fisher Scientific). Cell proliferation was examined using the Cell Proliferation ELISA BrdU kit (Roche, Basel, Switzerland) after incubation with bromodeoxyuridine (BrdU) for 6 hours. Cytotoxicity was measured using the Cytotoxicity Detection KitPLUS (lactate dehydrogenase [LDH] release assay; Roche). Selected cell survival assays used the following chemicals: NMDA (synthetic NMDAR agonist, 100 μM), L-glutamate (main NMDAR agonist, 500 μM; both from Sigma–Aldrich), glycine (NMDAR co-agonist, 300 μM; VWR International, Radnor, Pennsylvania, United States), memantine (NMDAR antagonist, 100 μM; Sigma–Aldrich), and cytarabine (0.1 μM; Cayman Chemical, Ann Arbor, Michigan, United States).

The human glioblastoma cell line, U87-MG was bought from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and supplemented with 10% fetal bovine serum at 37°C in a humidified 5% CO₂ incubator. Although one research published in Science Translational Medicine revealed that glioma cell line U87-MG from ATCC was likely to be a bona fide human glioblastoma cell line of unknown origin (30 (link)), there was a research also declared that studies of U87 still reflected brain-cancer biology and didn't need to be tossed out (31 (link)). So, we still used the U87-MG cell line to study the glioblastoma just like this research (32 (link)) Chloroquine (CQ) was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Bevacizumab was obtained from Roche Diagnostics (Basel, Switzerland). Anti-Bim, anti-Bcl-2, anti-Bax, anti-survivin, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-PARP, anti-LC3B-I, anti-LC3B-II, anti-SQSTM1 (p62), anti-Akt, anti-p70S6K, anti-mTOR, anti-GAPDH, anti-p-Akt (T308), anti-p-Akt (S473), anti-p-p70S6K (T389) and anti-p-mTOR (S2448) antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA). MTT kit was from Thermo Fisher Scientific, Inc. Annexin V/PI kit was from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

The SK-N-AS cells were cultured in 96-well plates with a seeding density of 500000 cells per well and transfected as previously described. Following transfection, the cells were starved with DMEM containing 0.1% FBS and incubated for 48 h, followed by GDNF treatment or no treatment. Based on the manufacturer's guide in the MTT kit (Thermo Fisher Scientific; V13154), the medium from each well was removed and replaced with 100 µl of fresh 1% FBS DMEM. Away from the light, 10 µl of the 12 mM MTT stock solution was added into each well and incubated at 37 °C for 4 h. After MTT incubation, 85 µl of medium was removed from each well, and 50 µl of DMSO was added to each well, mixed thoroughly with a pipette, and incubated at 37 °C for 10 min. Thereafter, the absorbance was read at 540 nm. The caspase-3/7 assay was conducted according to the manufacturer's guide (G8090; Promega), and the activity of caspase-3/7 was measured using a microplate reader at an excitation wavelength of 499 nm and an emission wavelength of 521 nm.