FAM-FLICA® Caspase-8 檢測試劑盒

使用 FLICA Caspase-8 Assay Kit 檢測 caspase-8 活性。這種體外測定採用螢光抑製劑探針 FAM-LETD-FMK 來標記活細胞中的活性 caspase-8 酶。使用螢光顯微鏡、螢光盤式分析儀或流式細胞儀分析樣品。

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 FAM FLICA Caspase-8 assay probe allows researchers to assess caspase-8 activation. The FLICA reagent FAM-LETD-FMK enters each cell and irreversibly binds to activated caspase-8. Because the FAM-LETD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-LETD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the active caspase-8 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 included in the kit. Unfixed samples may also be analyzed with Propidium Iodide or Hoechst 33342 to detect necrosis or changes in nuclear morphology, respectively.

Reagent Name FAM-LETD-FMK

Target Caspase-8

Excitation / Emission 488 nm / 530 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 10X 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 (e.g., add 10 μL to 290 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, Propidium Iodide, 7-AAD, or an antibody.

9. If desired, fix cells.

10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLICA excites at 492 nm and emits at 520 nm.

****If working with adherent cells, please see the manual for additional protocols.

Kit Contents

Kit #99: 25 Tests FLICA Caspase-8 Reagent (FAM-LETD-FMK), 1 vial, #656 10X Apoptosis Wash Buffer, 15 mL, #635 Fixative, 6 mL, #636 Propidium Iodide, 1 mL, #638 Hoechst 33342, 1 mL, #639 Kit Manual

Citations (3)

Lima, TS;Mallya, S;Jankeel, A;Messaoudi, I;Lodoen, MB. Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases. mBio. 2021 Jan 26; 12(1). doi: 10.1128/mBio.02031-20. Full Article Suzuki T, Kohyama K, Moriyama K, Ozaki M, Hasegawa S, Ueno T, Saitoe M, Morio T, Hayashi M, Sakuma H. Extracellular ADP augments microglial inflammasome and NF-κB activation via the P2Y12 receptor. Eur J Immunol. 2019 Sep 24. doi: 10.1002/eji.201848013. [Epub ahead of print]. https://onlinelibrary.wiley.com/doi/abs/10.1002/eji.201848013. Abstract Gornowicz A, Pawłowska N, Czajkowska A, Czarnomysy R, Bielawska A, Bielawski K, Michalak O, Staszewska-Krajewska O, and Kałuża Z. Biological evaluation of octahydropyrazin[2,1-a:5,4-a’]diisoquinoline derivatives as potent anticancer agents. Tumour Biol. 2017. Jun;39(6):1010428317701641. doi: 10.1177/1010428317701641. Full Text.