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3. Apoptosis Research

for semi-quntifying apoptotic cells in culture cells using TUNEL
Apoptosis Screening Kit wako
Wako Cat. No. 296-60001 (96 tests)
Keep at -20°C
Apoptosis Screening Kit Wako is designed to semi-quantify apoptotic cells in culture cells grown in a microtiter plate using the TUNEL (Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick en labeling) procedure. Fluorescein-labeled fragmented DNA by the TUNEL procedure is immunochemically quantifi ed by horseradish peroxidase (POD)-conjugated antibody and a chromogenic substrate on a microplate. This kit provides all the essential reagents for the assay.
1. Rapid detection can be performed in 3 hours.
2. Tedious preparations of various reagents are not needed.
[Kit Contents]
1) Fixation Solution 1 vial x 640 µL
2) Permeabilizing Solution1 vial x 19.2 mL
3) TdT 1 vial x 20µL 1 vial x 20 µL
4) TdT Substrate Solution1 vial x 4.8 mL
5) Hydrogen peroxide 1 vial x 340 µL
6) 500x POD-conjugated Antibody 1 vial x 20 µL
7) Antibody Diluent1 vial x 9.6 mL
8) Chromogenic Substrate5 tablets x 63 mg
9) Chromogenic Substrate Buff er1 vial x 10 mL
10) Stop Solution1 vial x 9.6 mL
11) Sterilized Microtiter Plate (96 wells)1 plate

HL-60 (104 cells) were seeded in each well and 1µg/mL of actinomycin D was added every one hour. The plate was incubated sequentially and the absorbance was measured. Then, the detection procedure according to the package insert was carried out. Cell death rate was calculated by morphologic observation of the cells under a microscope and the correlation between the rate and the absorbance was examined.
Apoptosis Inducer
Camptothecin, 98.0+% (HPLC)
Wako Cat. No. 038-18191 (100 mg); 034-18193 (500 mg)
Keep at 2~10°C <for Biochemistry>
An alkaloid contained in Nothapodytes foetida and Camptotheca acuminata. It has a quinoline skeleton but, biosynthetically, it is a resembling compound of indole alkaloid.
This product is a reversible inhibitor of DNA topoisomerase I and it binds to topoisomerase-DNA complex leading to stabilization. It exhibits antileukemic and antitumor activities and inhibits activation of HIV-1 by Tat. It exhibits cytotoxicity to tumorigenetic cells but not to nontumorigenetic cells. It also induces apoptosis in HL-60 cells and mouse thymus cells.
Anticancer irinotecan (Topotecin) is one of its derivatives.
Apoptosis Inducing Bioprobe
Cytotorienin A, from Streptomyces sp.
039-18241 (100 µg)
Keep at -20°C <for Biochemistry>

A unique bioprobe, cytotrienin A induces apoptosis (or programmed cell death) in human promyelocytic leukemia HL-60 cells at a low concentration (10 ng/mL).
Solubility : Soluble in methanol (0.1 mg/mL)

1) Kakeya, H., Zhang, H., Kobinata, K. Onose, R., Onozawa, C., Kudo T. and Osada, H., "Cytotrienin A, a Novel Apoptosis Inducer in Human Leukemia HL-60 Cells", J. Antibiotics, 50 (4), 370-372 (1997)
2) Zhang, H., Kakeya, H. and Osada H., "Novel Triene-ansamycins, Cytotrienins A and B, Inducing Apoptosis on Human Leukemia HL-60 Cells", Tetrahedron Letters, 38 (10), 1789-1792 (1997)
3) Kakeya, H., Onose, R., and Osada, H., "Caspase-mediated Activation of a 36-kDa Myelin Basic Protein Kinase during Anticancer Drug-induced Apoptosis", Cancer Research, 58, 5888-4894 (1998).
4) Watabe, M., Kakeya, H., Onose, R., and Osada, H., "Activation of MST/Krs and c-Jun N-terminal Kinases by Diff erent Signaling Pathways during Cytotrienin A-induced Apoptosis", J. Biol. Chem., 275, 8766-8771 (2000).
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Wako Product Update Bio-No.1 [ page. 12 ]