SR-FLICA® Caspase-3/7 檢測試劑盒

使用 SR-FLICA® 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 SR-DEVD-FMK 來標記活細胞中的活性 caspase 3 和 7 酶。使用螢光顯微鏡、螢光盤式分析儀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 and tissues. The FLICA reagent SR-DEVD-FMK enters each cell and irreversibly binds to activated caspase 3 and 7. Because the SR-DEVD-FMK FLICA reagent becomes covalently coupled to the active enzymes, it is retained within the cell, while any unbound SR-DEVD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining 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 a fluorescence plate reader, 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 33342 to detect changes in nuclear morphology.

Reagent Name SR-DEVD-FMK

Target Caspase-3/7

Excitation / Emission 550-580 nm / 590-600 nm

Method of Analysis Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader

Sample Type Cell culture

Storage 2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls.

2. Dilute Apoptosis Wash Buffer 1:10 with diH20.

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. For example, to stain at 1:30, add 10 µL to 290 µL of cultured cells. To stain at 1:60, add 5 µL to 295 µL of cultured cells.

6. Incubate approximately 1 hour.

7. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells.

8. If desired, label with additional stains, such as Hoechst, 7-AAD, or an antibody.

9. If desired, fix or embed cells.

10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. SR-FLICA excites at 550-580 nm and emits at 590-600 nm.

Kit Contents

Kit #931: 25 Tests FLICA Caspase-3/7 Reagent (SR-DEVD-FMK), 1 vial, #678 10X Apoptosis Wash Buffer, 15 mL, #635 Fixative, 6 mL, #636 Hoechst 33342, 1 mL, #639 Kit Manual