Mtt cell proliferation and cytotoxicity assay kit

PLGA (lactide:glycolide 50:50; Mw 38,000–54,000) and Poly vinyl alcohol (PVA, P875084) were purchased from MACKLIN (Shanghai, China). Astaxanthin (AST, SML0982) was purchased from Sigma-Aldrich (St Louis, MO, USA). MTT Cell Proliferation and Cytotoxicity Assay Kits were purchased from Beyotime (Shanghai, China). Dichloromethane and Ethanol were purchased from SINOPHARM (Beijing, China).

MTT cell proliferation and cytotoxicity assay kit (Beyotime Biotechnology, Shanghai, China) was used in this study for the cell proliferation assay of PBMCs stimulated by LPS and PHA. The human PBMCs were resuspended to a concentration of 2 × 10⁴ cells/mL. A 100 μL aliquot containing 2 × 10³ cells was added immediately to each well of a 96-well flat bottom microtitre plate in triplicate. LPS or PHA were added to the wells to the final concentration of 100 ng/ml (LPS) and 10 μg/ml (PHA). After a period of 72 h incubation at 37 °C in 5% CO₂, PBMCs proliferation was assayed with MTT kit.

Three human HCC cell lines, HepG2, SMMC-7721, and HuH-7 were infected with NDV at MOI = 0.01. The viability of cells at different time points was assessed by MTT cell proliferation and cytotoxicity assay kit (Beyotime Institute of Biotechnology, Haimen, China) with three repetitions for each sample. Absorbance at 570 nm was determined with BIO-TEK microplate readers (BioTek Instruments, Winooski, VT).

For cell viability analysis, MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Biotechnology, Suzhou, China) was used according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates (3 × 10³/well) and treated with various concentrations of β-Thujaplicin. At the indicated time points, the old medium was replaced with 100 μL of fresh medium containing 10 μl of MTT solution. After 4 h of incubation at 37 °C, formazan crystals were solubilized with 100 μl of formazan dissolution reagent . The absorbance levels for each sample at 570 nm were measured using a microplate reader (Bio-Tek, Winooski, VT, USA). The data were duplicated six times.

β-Thujaplicin were purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of β-Thujaplicin at 100 mM was prepared in DMSO (Sigma, St. Louis, MO, USA) and stored at −20 °C. Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were obtained from Basal Media Biotechnology (Shanghai, China). AO/EB staining kit was purchased from Sangon Biotech (Shanghai, China). MTT cell proliferation and cytotoxicity assay kit, BCA protein assay kit, and apoptosis analysis kit were purchased from Beyotime Biotechnology (Suzhou, China). Annexin V-FITC/PI Apoptosis detection kit was purchased from KeyGen Biotech (NanJing, China). Crystal violet solution, CQ, and EBSS were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used in the concentration indicated before. LY3214996 and RAPA were purchased from Selleck Chemicals (Texas, USA). SB203580 was purchased from MedChemExpress (New Jersey, USA). Ad-GFP-LC3 adenovirus (Hanbio Biotechnology, Shanghai, China) was used to detect autophagosomes. The primary antibodies GAPDH, p-Akt, Akt, p38, p-p38, JNK, p-JNK, ERK, p-ERK, p62, LAMP1, LC3B, cleaved PARP1, cleaved caspase-3, caspase-3, Bax, and Bcl-2 were afforded by Cell Signaling Technology (Beverly, MA, USA).

The pUSE-PTHLH plasmid or siRNA was transfected when cells were grown to 80% confluency using NanoFectin Transfection Reagent (Shanghai ExCell Biology, Shanghai, China). Twenty-four hours later, the cells were cultured with serum-free M199 medium for another 24 h.
Proliferation of cells was quantified by MTT staining (Berridge and Tan, 1993 (link)) at 0, 24, 48, and 72 h after cell transfection using an MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Beijing, China). The MTT solution (5 mg/mL) that was assessed and incubated for 4 h at 37°C was added to formazan lysis buffer for a further 4 h incubation until all the formazan dissolved. Cell number was evaluated using an ELx808 Absorbance Reader at 570 nm.