Reagents:
Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI), Dulbecco’s Minimal Essential medium (DMEM), retinoic acid, 4, 6-diamidino-2-phenylin- dole (DAPI) Penicillin G, TEMED, triton X-100, glycine, sodium bicarbonate, tween 20, Phosphate buffered saline (PBS), sodium fluoride, EDTA, Aβ1-42, sodium orthovanadate, paraformaldehyde, skimmed milk, Radioimmunoprecipitation assay buffer (RIPA), trypsin, sodium pyruvate, sodium dodecyl sulphate, Streptomycin sulphate, rapamycin, bovine serum albumin (BSA) dimethylsulfoxide (DMSO), ammonium persulfate, HEPES, glycerol, sodium chloride, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), , anti-LC3 and anti-SQSTM1 antibodies were purchased from Sigma-Aldrich. Lipofectamine, fetal bovine serum (FBS), Human Aβ1-42 kit, were obtained from Invitrogen. Aβ1-42-HiLyte FluorTM 555 were purchased from Anaspec. ECL-kit and PVDF membrane were purchased from Millipore. Bradford and protein marker ladder was obtained from Bio-rad. Antibodies were purchased from Cell Signalling Technology.
Cell culture:
Human microglial HMC3 cells were obtained from American Type Culture Collection (ATCC). HMC3 cells were grown in RPMI media and Human SHSY-5Y cells maintained in MEM media complemented with 100 mg/L streptomycin, 80 mg/L penicillin G and 10% FBS. These cells were grown in an incubator containing CO2 (5 %) and 95 % humidity. SHSY-5Y cells were differentiated into 5% FBS containing 10 mM retinoic acid for 7 days and neuronal markers were checked through western blot.
Western blot:
Microglial HMC3 cells grown into 60 mm dishes and were treated with betaine for 24 h at a concentration of 1, 5 and 10 mM. The cells were washed with PBS followed by lysis with RIPA bufffer having sodium orthovanadate, cocktail and sodium fluoride for 1 h. These samples were collected after centrifugation and proteins were estimated by bradford reagent followed by the standard curve by using BSA. Equal amount of proteins were loaded into each wells and were run for 2 hours in SDS page buffer at 100V. These samples were transferred into PVDF membrane at 100V for 2 h and the protein membranes were incubated into 5% skimmed milk for blocking. Primary antibodies were probed into each membrane and secondary antibodies were treated for 1 h followed by 5 min washing with PBST (0.3% Tween-20). The detection of antibodies were done by ECL Kit using X-ray.
Transient transfection of GFP-LC3 in HMC3 cells:
HMC3 cells were seeded on c coverslips and were transfected with GFP-LC3 plasmid (1 μg of plasmid+ Lipfectamine 20 μl per ml) for 24 h. The transfected cells were treated with betaine at a concentration of 1, 5 and 10 mM. for 24 h. These cells were fixed for 30 min with 5% paraformaldehyde and slides were prepared by using mounting media and analyzed through confocal microscope.
Amyloid beta clearance through confocal microscope:
HMC3 cells were seeded into coverslips and pre-treated with betaine at a concentration dependent manner for 12 h followed by the treatment of hilyte Aβ1-42 555 for 24 h and DAPI (1 mg/ml) for 15 min. Cells were fixed with 5% paraformaldehyde for 30 min and slides were prepared for confocal microscopy.
ELISA for amyloid beta clearance:
HMC3 cells were pre-treated with betaine at a concentration dependent manner for 12 hours and Ab1-42 for 24 h. Cells were washed, lysed and performed as suggested by the manufacturer instructions.
Transient transfection of LC3 in differentiated SHSY5Y cells:
SHSY5Y cells were differentiated into retinoic acid for 7 days and transfected with siLC3 for 24 h. After 24 h, cells were pre-treated with betaine followed by Ab1-42 for 48 h. Before 4 h of termination MTT were added followed by DMSO into each well. The absorbance was taken at 570 nm on plate reader. The untreated controls were taken as 100% and the comparison were made between control vs amyloid beta and amyloid beta vs control.
Neuroprotection:
Differentiation of human SHSY-5Y cells was obtained by 5% FBS and 10 mM retinoic acid for 7 days. After differentiation, betaine was pre-treated into SHSY-5Y for 24 h and Ab1-42 (10 mM) treatment for 48 hours. MTT dye (2.5 mg/ml) was incubated into each well. After 4h, DMSO was added into each well. The absorbance was taken at 570 on plate reader. The untreated controls were taken as 100% and the treated groups were compared with control.
Statistical Analysis:
Mean±SD of three independent experiments is shown here and students T-test were performed for comparison between groups. P<0.05 was considered as statistical significant.