Sodium Benzoate Cell culture personal notes and procedures

Cell culture materials such as HBSS, L-Glutamine, 0.05% trypsin, and DMEM/F-12 were purchased from Invitrogen and fetal bovine serum (FBS) was obtained from Atlas Biologicals. All other molecular biology chemicals were purchased from Sigma. NaB, was acquired from Sigma. Human Neuroblastoma. Sh-SY5Y modification were obtained from Sigma, and were prepared as followed (9,10). The Institutional Review Board of the Rush University Medical Center approved all experimental protocols. About, 8-15 week-old culture cells were used, and had confluent growth on 24 wells and treatment for different time frames to create a greater resolution of RNA production based on time. Treatment dilutions of NaB and cinnamic acid. Time treatments intervals and varying concentrations were used for NaB and cinnamic acid. For NaB concentrations we used a control of zero concentration then 50µM, 100µM, 150µM, and 200µM; and time treatment of 30 min, 60min, 120min, 180min, and 240min with 100µM NaB concentration. For cinnamic acid concentrations we used a control of zero
concentration then 50µM, 100µM, 200µM, 300µM, and 500µM; and time treatment of30 min, 60min, 120min, 180min, and 240min with 300 µM cinnamic acid.RT-PCR and real time qPCR. RNA from neuroblastomas were isolated using Qiagen RNA-Easy kit and their protocol’s were used. cDNA was then synthesized using dNTP, 0.1M DTT, oligo-dT, RNaseOUT, M-MLV RT, and 5x FS Buffer all from Invitrogen which was then amplified with Promega’s PCR Master Mix with the primers from Invitrogen.CREB, (antisense) 5?-TGC CAA GCC AGT CCA TTC TCC AC-3?; (sense) 5?-TGC AGC TGC CAC TCA GCC GGG-3?.BDNF, (antisense) 5?TGT CAC ACA CGC TCA GCT CCC C-3; (sense) 5?-AGG CAA CTT GGC CTA CCC AGG TGT G-3?,NT-3, (antisense) 5?-GTC ATC AAT CCC CCT GCA ACC GTT T -3?; (sense) 5?-GAG…