Pyroptosis/Caspase-1 檢測，綠色

Pyroptosis/Caspase-1 檢測試劑盒利用 FLICA 技術來檢測 caspase-1 的激活。使用該試劑盒，研究人員可以輕鬆評估焦亡細胞並利用尼日利亞菌素作為陽性對照。使用螢光顯微鏡、螢光盤式分析儀或流式細胞儀分析綠色光訊號。

Background Inflammatory caspases, such as caspase-1, play a critical role in the mechanism to trigger the lytic programmed cell death known as pyroptosis. ICT’s Pyroptosis/Caspase-1 Assay, Green can be used by researchers to assess caspase-1 activity in pyroptotic cells and utilize nigericin as a positive control. The FLICA reagent FAM-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the FAM-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-YVAD-FMK reagent diffuses out of the cell and is washed away. The remaining green 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 a fluorescent plate reader, fluorescence microscopy, or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 24 hours using the fixative. Unfixed samples may also be counter-stained with Propidium Iodide, 7-AAD, or Hoechst (included in the kit). Nigericin induces a net decrease in intracellular levels of potassium, crucial for activation of caspase-1. In pyroptosis experiments, nigericin can be used as a positive control to generate a robust caspase-1 activation response in a variety of cell lines.

Reagent Name FAM-YVAD-FMK

Target Caspase 1

Excitation / Emission 488 nm / 430 nm

Method of Analysis Flow cytometry, Fluorescence microscope, fluorescence plate reader

Sample Type Cell culture, tissue

Storage Nigericin at ≤ -20°C other components at 2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls.

2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O.

3. Reconstitute FAM-FLICA with 50 µL DMSO.

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

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

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 33342, 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 excited at 492 nm and emits at 520 nm.

Kit Contents

Kit# 9145: 25-50 Tests FLICA Caspase-1 Inhibitor Reagent (FAM-YVAD-FMK), 1 vial, #655 10X Cellular Wash Buffer, 15 mL, #6164 Fixative, 6 mL, #636 Hoechst 33342, 1 mL, #639 Nigericin, 0.5 µmoles, #6698 Kit Manual

Citations (2)

Samukawa, S;Yoshimi, R;Kirino, Y;Nakajima, H. The PRY/SPRY domain of pyrin/TRIM20 interacts with ?2-microglobulin to promote inflammasome formation. Scientific reports. 2021 December 8; doi: 10.1038/s41598-021-03073-6. Text Chen, A;Chen, Z;Zhou, Y;Wu, Y;Xia, Y;Lu, D;Fan, M;Li, S;Chen, J;Sun, A;Zou, Y;Qian, J;Ge, J. Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome activation. Cell Death & Disease. 2021 Jan 12; 12(1)78. doi: 10.1038/s41419-021-03389-1. Full Article