![background image](http://html.mh-extra.com/html/takara/mk301/mk301_manual_814465005.webp)
3. Dilute the cell lysate step-wise with physiological saline and add the prescribed
amount (in the range of 5 - 50 μ l) to each well of the provided 96-well plate.
Following this step, the procedure is the same as in (1) 4. - 5.
(3) For blood samples
1. Add 5 - 50 μ l of blood samples (serum, plasma) to each well.
Note 1) Sample should be diluted with Extraction solution [2].
Following this step, the procedure is the same as in (1) 4. - 5.
VIII. Application Examples
(1) Measurement of Alkaline Phosphatase (ALP)
This kit was used to measure ALP activity in cultured cells derived from human
small intestine. At the same time, ALP staining was conducted using the fixation
solution and insoluble substrate BCIP/NBT included in the TRACP & ALP Double-
stain Kit (Cat. # MK300) .
< Method >
Suspension of human small intestine cells (Intestine 407) that were serially cul-
tured in 10 % FCS/RPMI1640 medium was added to the first row of a 96-well plate.
The plate was prepared up to the 11th row by 2-fold dilution (No. of cells:1 x 10
4
in row 1, 5 x 10
3
in row 2, and thereafter decreasing by half; row 12 was blank) ,
then further cultured for 2 days (volume:100 μ l/well) . After culturing, ALP stain-
ing was conducted with insoluble substrate BCIP/NBT on columns A and B, and
ALP activity was measured in columns C - F using this kit and Procedure (1) . Due
to solubilization, the volume of extraction solution added to columns C - F varied
from 5 to 50 μ l (5 μ l in column C, 10 μ l in column D, 20 μ l in column E, 50 μ l
in column F) . Enzymes reactions were conducted for 60 minutes at 37 ℃ .
< Results >
Alkaline phosphatase activity is extremely strong in the small intestine cells, and
the correlation of activities and number of cells were confirmed using this kit.
The detection sensitivity for human small intestine cells (Intestine 407) under
the conditions of this activity measurement was 10 cells/assay. It was confirmed
that even though the volume of extraction solution added varied in the range of
5 to 50 μ l/well, there was little influence on the enzyme reactions. There is no
problem in varying the volume of extraction solution within a fixed range if the
cells are solubilized at a sufficient volume.
5
TRACP & ALP Assay Kit
Cat. #MK301
v0804
URL:http://www.takara-bio.com