Simulative Evaluation of Taurine Against Alopecia Caused by Stress in Caenorhabditis elegans - Advances in Experimental Medicine and Biology - page 253

25.2 Methods

25.2.1 Synchronous Culture Conditions

and Induction of ER Stress

Wild type of C . elegans N2 was cultured at 25°C on the nematode growth medium
(NGM) according to the standard method (Stiernagle 2006 ; Szewczyk et al. 2003 ) .
To induce ER stress, worms were grown for 3 h on media containing tunicamycin at
10 mg/ml. They were further incubated with various concentrations of anti-alopecia
agents. The anti-alopecia agents used in this experiment were astressin-B, finasteride,
and taurine which were all purchased from Sigma (St. Louis, USA). The anti-alopecia
agents were delivered into the media as the final concentration of 10 or 100 m g/ml.
The stress conditions were assessed by examining the stress protein marker expression.
The expression of hsp-70 was detected according to the standard immunoblotting
procedures. The antisera against hsp-70 were purchased from Santa Cruz
Biotechnology (Santa Cruz, USA) and diluted as 1,000 times for immunoblotting in
the present experiments.

25.2.2 Measurement of Worm's Vital Signs: Lifespan Extension,

Progeny Number, and Mobility Restoration

The effect on life length extension was estimated using the method of Hyun et al.
( 2008 ). As a brief description, worms were initially destroyed except the eggs by
bleaching and ten eggs were incubated on NGM supplemented with OP50 at 25°C
until worms reached the young adult stage. Fifty worms were then transferred onto
plates containing 10 mg/ml of tunicamycin, where they were incubated for 3 h. They
were subsequently transferred to media containing 10 or 100 mg/ml of astressin-B,
finasteride, or taurine. Live worms were counted daily. Worms were excluded from
counting if they failed to react to a stimulus with a platinum wire. The lifespan
extension effect was calculated as in a rate of rescuing worms from the chemical
stress. The rescue rate (RR, %) was calculated according to the following formula:
RR(%) = (A−B)/(C−B) X 100(%), where A refers to offspring number with each
drug treatment after Tun, B to offspring number with Tun-only treatment, and C to
offspring number with drug-free treatment.
In order to evaluate the effect on offspring number, the number of eggs was
counted daily and standardized per number of adult after the anti-alopecia treat-
ment under the ER stress conditions. The adults were selected for visual consis-
tency and removed to a fresh plate with 10 mg/ml of tunicamycin. After subsequent
3 h incubation on the Tun media, they were further treated with anti-alopecia
agents at 10 or 100 mg/ml. The number of fertilized eggs and larvae was counted
and standardized as a rate of increase on 2, 3, and 4 days after the beginning of
the culture.