FLISP FAM-Leu-CMK 絲氨酸蛋白酶檢測試劑盒

FLISP 試劑盒用在體外定量細胞內胰凝乳蛋白酶樣絲氨酸蛋白酶活性。糜蛋白酶靶向 FLISP 探針與糜蛋白酶樣蛋白酶的活性催化位點相互作用，因此具有更多活性糜蛋白酶樣酶活性的細胞比未上調的細胞具有更大的螢光潛力。使用流式細胞儀、螢光顯微鏡或螢光盤式分析儀分析您的樣品。

Background Because of their supporting role in the apoptotic process, serine protease activity will be greater in apoptotic cell populations compared to healthy cells of the same type. Quantitate intracellular chymotrypsin-like serine protease activity in vitro with ICT’s FLISP kits. If there is an active chymotrypsin-like enzyme inside the cell, it will covalently bind with the FLISP inhibitor, in this case FAM-Leu-CMK (FLCK), and retain the green fluorescent signal inside the cell. Cells containing lower concentrations of chymotrypsin-like enzyme activity will retain a lower level of fluorescence compared to cells containing higher concentrations of this effector enzyme component. FLISP may be used in combination with FLICA to discriminate serine protease activity from caspase activity in the same cell. Analyze results using a flow cytometer, fluorescence microscope, or fluorescence plate reader.

Reagent Name FFCK

Target Chymotrypsin-like enzymes

Excitation / Emission 488 nm / 520 nm

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

Sample Type Cell culture, tissue

Storage FLISP at ≤ -20°C, other components at 2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls

2. Dilute cellular wash buffer 1:10 with diH2O

3. Reconstitute FLISP with 50 µL DMSO

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

5. Add 10 µL FLISP to each sample (~500 µL aliquot of cultured cells)

6. Incubate ~ 1 hour.

7. Remove media and wash cells: add wash buffer and spin cells (twice); or add fresh media and incubate 1 hour

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

9. If desired, fix or embed cells

10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLISP excites at 488 nm and emits at 520 nm.

Kit Contents

Kit# 949: 25 Tests FAM-Leu-CMK, 1 vial, #6148 Hoechst Stain, 1 mL, #639 Propidium Iodide, 1 mL, #638 10X Cellular Wash Buffer, 15 mL, #6164 Fixative, 6 mL, #636 Kit Manual