< Results >
The bone marrow cell culture produces numbers of naturally differentiated osteo-
clast-like TRACP-positive cells, and their TRACP activity could be measured using
this kit. The table below shows the numbers of cells at the start of culturing and
TRACP activity as absorbency at 405 nm. Similar levels of activity were detected in
the live cells to which substrate was directly added without extraction (final sub-
strate concentration 12.5 mM) and in cells on which extraction was conducted (final
substrate concentration 4.1 mM) , and results that correlated with the numbers of
cells were obtained.
No. of cells (cells/well)
7 × 10
5
3.5 × 10
5
1.75 × 10
5
8.76 × 10
4
4.38 × 10
4
2.19 × 10
4
Substrate directly added
1.701
1.471
0.747
0.295
0.156
0.120
Extracted
1.754
1.880
0.781
0.246
0.150
0.125
1.1 × 10
3
5.5 × 10
2
2.25 × 10
2
112
56
0
0.105
0.100
0.104
0.102
0.100
0.100
0.112
0.111
0.112
0.107
0.105
0.108
(3) Measurement with Acid Phosphatase (ACP) Standards
A standard curve was made using ACP (Code 108227, Lot 93207721) from Roche
Diagnostics.
Distilled water was added to the reference standards to prepare 100 μ g/ml en-
zyme solutions, and a 2-fold dilution series was prepared using each enzyme buf-
fer.
Using 1 well of a 96-well plate for 1 reaction, 50 μ l of enzyme and 50 μ l of the
substrate solution
*
were mixed and reacted for 30 minutes at 37 ℃ . After adding
50 μ l of stop solution, absorbency at 405 nm was immediately measured using a
plate reader.
Note) In the case of acid phosphatase, color forms with the addition of stop solution.
*:Tartaric acid is not added to acid phosphatase substrate.
< Results >
In the reaction scales produced:
For ACP, concentrations were measurable from 1.5 μ g/ml or less, and actual lin-
earity was obtained in the range up to 0.5 μ g/ml.
7
TRACP & ALP Assay Kit
Cat. #MK301
v0804
URL:http://www.takara-bio.com