Taurine as a Marker for the Identification of Natural Calculus Bovis and Its Substitutes - Advances in Experimental Medicine and Biology - page 135

Table 15.1 Summary of the materials used in the study
No. Species Properties Collection site Collection year Sample name

1 Natural C . Bovis Powder Australia 2006 Aus-1

2 Natural C . Bovis Powder Australia 2001 Aus-2

3 Natural C . Bovis Powder Argentine 2001 Argentine

4 Natural C . Bovis Powder Brazil 2001 Brazil

5 Natural C . Bovis Powder Guatemala 2001 Guatemala

6 Natural C . Bovis Powder Mexico 2001 Mexico

7 Natural C . Bovis Powder Kenya 2001 Kenya

8 Natural C . Bovis Clod China 2007 China

9 Natural C . Bovis Clod India 1978 India

10 Arti fi cial C . Bovis Powder China 2007 Art-1

11 Arti fi cial C . Bovis Powder China 1971 Art-2

12 In vitro cultured C . Bovis Clod China, Anhui 2009 Cul-1

13 In vitro cultured C . Bovis Clod China, Hubei 2009 Cul-2

15.2 Methods

15.2.1 Sample Preparation

Thirteen samples of C . Bovis were collected from various locations as shown in
Table 15.1 . Dr. K. Takahashi purchased the samples named “China” and “Art-1”
from local stores in Shenyang, while “India” and “Art-2” are historical specimens
stored in the Museum of Osaka University, Japan. The rest of the specimens were
purchased from Tochimoto Tenkai-Do (Osaka, Japan). Seventy-five milligram of
each sample was extracted with 1 ml DMSO and then 0.5 ml water was added.
Samples were vortexed, and sonicated for 5 min before centrifugation at 12,000 × g
for 20 min at 4°C. The supernatant was collected and stored at −20°C.

15.2.2 Evaluation of Cell Viability

Primary cardiac fibroblast cultures were prepared from 1-day-old Wistar rats as
described previously (Takahashi et al. 2002 ). The cells were seeded in a 96-well plate
after a few days' incubation in serum-containing culture medium, Dulbecco's modified
Eagle's medium/F-12 (Dainippon Pharmaceutical Co., Ltd.) (1:1 vol/vol) supple-
mented with newborn calf serum (GIBCO; 10%, heat-inactivated). After 24-h incuba-
tion in serum-containing culture medium, cells were transferred to serum-free medium.
After 24-h incubation, cells were treated with 0.25 mg/ml of Aus-1, Art-1, Cul-1, and
Cul-2 for 1 h. Following removal of the sample solution, the cells were then incubated
in serum-free medium (100 ml) with CellTiter 96® aqueous one solution reagent
(Promega Corporation) (20 ml) for 3 h. Cell viability was defined as the ratio (expressed
as a percentage) of absorbance of treated cells to untreated cells at 490 nm.