Sodium Benzoate Cell culture personal notes and procedures

Sodium Benzoate Cell culture personal notes and procedures

  • Submitted By: renkaimo
  • Date Submitted: 05/07/2014 3:36 PM
  • Category: Science
  • Words: 352
  • Page: 2

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 100

µM NaB concentration. For cinnamic acid

concentrations we used a control of zero
concentration then 50µM, 100µM, 200µM,

300µM, and 500µM; and time treatment of

30 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...

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