FLICA 660 Caspase-1 檢測試劑盒

使用 FLICA 660 Caspase-1 檢測試劑盒檢測 caspase-1 活性。這種體外測定採用螢光抑製劑探針 660-YVAD-FMK 來標記活細胞中的活性 caspase-1 酶。使用螢光顯微鏡或流式細胞儀分析螢光信號。

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-1 probe allows researchers to assess caspase-1 activation. The FLICA reagent 660-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the 660-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound 660-YVAD-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-1 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-VAD-FMK

Target

Caspase 1

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 Cellular 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 (e.g. spike at 1:30 by adding 10 µL to 290 µL cultured cells).

6. Incubate approximately 1 hour.

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

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-1 Reagent (660-YVAD-FMK), 1 vial, #6323 10X Cellular Wash Buffer, 15 mL, #6164 Fixative, 6 mL, #636 Kit Manual

Citations (7)

Shen L, Yang Y, Ou T, Key CC, Tong SH, Sequeira RC, Nelson JM, Nie Y, Wang Z, Boudyguina E, Shewale SV, Zhu X. Dietary PUFAs attenuate NLRP3 inflammasome activation via enhancing macrophage autophagy. J Lipid Res. 2017 Jul 20. pii: jlr.M075879. doi: 10.1194/jlr.M075879. [Epub ahead of print]. Full Text. Moghaddas F, Llamas R, De Nardo D, Martinez-Banaclocha H, Martinez-Garcia JJ, Mesa-Del-Castillo P, Baker PJ, Gargallo V, Mensa-Vilaro A, Canna S, Wicks IP, Pelegrin P, Arostegui JI, Masters SL. A novel Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to Familial Mediterranean Fever. Ann. Rheum. Dis. 2017. Aug 23. pii: annrheumdis-2017-211473. doi: 10.1136/annrheumdis-2017-211473. [Epub ahead of print]. Abstract Swanson KV, Junkins RD, Kurkjian CJ, Holley-Guthrie E, Pendse AA, El Morabiti R, Petrucelli A, Barber GN, Benedict CA, Ting JP. A noncanonical function of cGAMP in inflammasome priming and activation. J. Exp. Med. 2017. Dec 4;214(12):3611-3626. doi: 10.1084/jem.20171749. Epub 2017 Oct 13. Abstract Mishra SK, Gao YG, Deng Y, Chalfant CE, Hinchcliffe EH, Brown RE. CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation. Autophagy. 2017. Nov 22:1-46. doi: 10.1080/15548627.2017.1393129. [Epub ahead of print]. Abstract Köse-Vogel N, Stengel S, Gardey E, Kirchberger-Tolstik T, Reuken PA, Stallmach A, Bruns T. Transcriptional Suppression of the NLRP3 Inflammasome and Cytokine Release in Primary Macrophages by Low-Dose Anthracyclines. Cells. 2019 Dec 28;9(1). pii: E79. doi: 10.3390/cells9010079. Full Text Martín-Nalda A, Fortuny C, Rey L, Bunney TD, Alsina L, Esteve-Solé A, Bull D, Anton MC, Basagaña M, Casals F, Deyá A, García-Prat M, Gimeno R, Juan M, Martinez-Banaclocha H, Martinez-Garcia JJ, Mensa-Vilaró A, Rabionet R, Martin-Begue N, Rudilla F, Yagüe J, Estivill X, García-Patos V, Pujol RM, Soler-Palacín P, Katan M, Pelegrín P, Colobran R, Vicente A, Arostegui JI. Severe Autoinflammatory Manifestations and Antibody Deficiency Due to Novel Hypermorphic PLCG2 Mutations. J Clin. Immunol. 20 Jul 15. doi: 10.1007/s10875-020-00794-7. Online ahead of print. Full Text Li H, Jiang W, Ye S, Zhou M, Liu C, Yang X, Hao K, Hu Q. P2Y14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages. Cell Death Dis. 2020 May 26;11(5):394. doi: 10.1038/s41419-020-2609-7. Full Text Question: FLICA 660 optimally excites at 660 nm and has a peak emission at 685-690 nm, and Propidium Iodide excites at 615nm. Can I use the FLICA 660 and the Propidium Iodide to detect pyroptosis by Flow cytometry?

Frequently Asked Questions

Question: FLICA 660 optimally excites at 660 nm and has a peak emission at 685-690 nm, and Propidium Iodide excites at 615nm. Can I use the FLICA 660 and the Propidium Iodide to detect pyroptosis by Flow cytometry? Answer: We have done some two-color panels pairing FLICA 660 with green emission fluors, however, we have not evaluated it alongside Propidium Iodide. That said, despite emission spectra overlap between Propidium Iodide and 660-YVAD-FMK I believe it should be possible to resolve the red vs far red fluors with appropriate compensation. I wanted to make you aware of Green Live/Dead Stain, ICT’s membrane impermeant green fluorescent vital stain for differentiating live and dead cells. This product performs similarly to Propidum Iodide, however, due to its green emission properties, it is compatible with our FLICA 660 products without the need for compensation.