FLICA 660 Caspase-3/7 檢測試劑盒

使用 FLICA 660 Caspase 3/7 Assay Kit 檢測 caspase 3 和 7 的活性。這種體外測定採用螢光抑製劑探針 660-DEVD-FMK 來標記活細胞中的活性 caspase 3 和 7 酶。使用螢光顯微鏡或流式細胞儀分析螢光訊號。

Background Caspases play important roles in apoptosis and inflammation. ICT’s FLICA assay kits are used by researchers seeking to quantitate apoptosis via caspase activity in cultured cells. The FLICA 660 Caspase 3/7 probe allows researchers to assess caspase 3 & 7 activation. The FLICA reagent 660-DEVD-FMK enters each cell and irreversibly binds to activated caspase 3 and 7. Because the 660-DEVD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound 660-DEVD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining far red fluorescent signal is a direct measure of the active caspase 3 and 7 enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by fluorescence microscopy or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative. Unfixed samples may also be analyzed with Hoechst stain to detect changes in nuclear morphology.

Reagent Name 660-DEVD-FMK

Target Caspase 3 and 7

Excitation / Emission 660 nm / 690 nm

Method of Analysis Flow cytometry, Fluorescence microscope

Sample Type Cell culture

Storage 2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls

2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH2O.

3. Reconstitute FLICA with 50 µL DMSO.

4. Dilute FLICA 1:5 by adding 200 µL PBS.

5. Add diluted FLICA to each sample at 1:30-1:60 (e.g. spike at 1:30 by adding 10 µL to 290 µL cultured cells).

6. Incubate approximately 1 hour.

7. Wash and spin cells three times.

8. If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.

9. If desired, fix cells.

10. Analyze with a fluorescence microscope or flow cytometer. FLICA 660 excites at 660 nm and emits at 680-690 nm.

Kit Contents

FLICA Caspase-3/7 Inhibitor Reagent (660-DEVD-FMK), 1 vial, #6344 10X Apoptosis Wash Buffer, 15 mL, #635 Fixative, 6 mL, #636 Kit Manual

Citations (2)

Palacios-Macapagal D, Connor J, Mustelin T, Ramalingam TR, Wynn TA, and Davidson TS. Cutting Edge: Eosinophils Undergo Caspase-1-Mediated Pyroptosis in Response to Necrotic Liver Cells. J. Immunol. 2017. Aug 1;199(3):847-853. doi: 10.4049/jimmunol.1601162. Epub 2017 Jun 26. Abstract. Peng R, Zhang W, Zuo Z, Shan Y, Liu X, Tang Y, Yu Z, Wang L, Cong Y. Dimethyl sulfoxide, a potent oral radioprotective agent, confers radioprotection of hematopoietic stem and progenitor cells independent of apoptosis. Free Radic Biol Med. 2020 Mar 25;153:1-11. doi: 10.1016/j.freeradbiomed.2020.03.021. [Epub ahead of print]. Full Text