The staining of ALP was carried out as follows.
1 x 10
4
cells / well
(2) Measurement of Tartrate-resistant Acid Phosphatase (TRACP)
< Method >
Bone marrow cells derived from the femur of 16-week-old rabbit were suspended
in 10 % FCS/RPMI1640 medium. This cell suspension was added to the first row of
a 96-well plate, and the plate was prepared up to the 11th row by 2-fold dilution
of the same culture (No. of cells:7 x 10
5
in row 1, 3.5 x 10
5
in row 2, and thereafter
decreasing by half in the same manner; row 12 was blank) , at a volume of 100 μ l/
well, and culturing was initiated. A further 100 μ l of medium was added because
adhesive cells had become prominent after 3 days, and culturing was continued.
Eight days after seeding, TRACP activity was measured using the kit according to
procedure (1) .
The degree of substrate color formation was compared when substrate solution
was directly added to the adhesive cells without extraction procedure and when
they were solubilated with 25 μ l of extraction solution. 50 μ l of substrate solution
for TRACP was used, and enzyme reactions were conducted for 60 minutes at 37 ℃ .
Plate row
1
2
3
4
5
6
No. of cells
(cells/well)
1 × 10
4
5 × 10
3
2.5 × 10
3
1.25 × 10
3
6.25 × 10
2
3.13 × 10
2
A405 Plate Column
C
5 μ l
*
2.275
2.271
1.732
0.920
0.581
0.329
D
10 μ l
*
2.298
2.263
1.957
1.082
0.719
0.373
E
20 μ l
*
2.289
2.273
1.901
1.101
0.617
0.369
F
50 μ l
*
2.236
2.214
1.964
1.287
0.744
0.341
*:Volume of extraction solution added
7
8
9
10
11
12
1.56 × 10
2
7.8 × 10
1
3.9 × 10
2
20
10
0
0.239
0.180
0.160
0.158
0.154
0.145
0.252
0.200
0.176
0.154
0.158
0.148
0.228
0.192
0.168
0.162
0.152
0.145
0.234
0.175
0.161
0.152
0.157
0.145
6
TRACP & ALP Assay Kit
Cat. #MK301
v0804
URL:http://www.takara-bio.com