Name: SR101 FLISP SFCK Serine Protease Assay
SKU: 951, 952
Availability: InStock

Serine proteases are a family of proteolytic enzymes defined by the presence of a serine residue at the active center of the enzyme. Activated serine proteases play major roles in several different functions including: apoptosis, markers of tumor malignancy, diagnostic and prognostic indicators of breast carcinomas and neck and head carcinomas. Serine protease activity is also altered in a variety of other cell-mediated diseases related to transplant rejection and infections. The most characterized enzymes of this type are trypsin and chymotrypsin. All living cells have a base level of chymotrypsin-like enzyme activity which will vary with the physiological state of the cell as well as by cell type. 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 cell type. Activation of caspases is upstream and likely a prerequisite for activation of serine proteases. FLISP can be used in combination with FLICA to discriminate serine protease activity and caspase activity in the same cell.

The FLISP reagent SR101-Phe-CMK enters each cell and binds to activated chymotrypsin-like serine proteases. Because the SR101-Phe-CMK FLISP reagent becomes covalently coupled to the active enzymes, it is retained within the cells, while any unbound SR101-Phe-CMK FLISP reagent diffuses out of the cell and is washed away. The remaining red fluorescent signal is a direct measure of the active chymotrypsin-like serine protease activity present in the cell at the time the reagent was added. Cells that contain the bound FLISP can be analyzed by a fluorescence plate reader, fluorescence microscopy, or flow cytometry. Cells labeled with the FLISP reagent may be read immediately or preserved for up to 16 hours using the fixative included in the kit. Samples may also be analyzed with Hoechst 33342 (included in the kit) to detect changes in nuclear morphology.

1. Prepare samples and controls.
2. Dilute 10X 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 three times: add 1X Cellular Wash Buffer and spin cells.
8. If desired, label with additional stains, such as Hoechst 33342, or an antibody.
9. If desired, fix cells.
10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. SR101-FLISP excites at 586 nm and emits at 605 nm.

Suspension cells were treated with an agent to induce serine protease activity, (bottom), or DMSO, a negative control (top), then incubated with ICT’s red serine protease inhibitor probe, SR101-FLISP, washed, and analyzed with a flow cytometer. The experimental treatment induced serine protease activity in 56.3% of the experimental cells (M2, bottom right), whereas the negative control treatment exhibited serine protease activity in only 13.5% of the cell population (M2, top right). This is a ratio of 4:1 (Dr. Brian W. Lee & Sally Hed, ICT).

• Reagent name: SR101-Phe-CMK (SFCK)
• Target: Chymotrypsin-like enzymes
• Excitation/Emission: 586 nm / 605 nm
• Method of Analysis: Flow cytometry, Fluorescence microscope, Fluorescence plate reader
• Types of Samples: Cell culture
• Storage: FLISP at ≤ -20°C, all other kit components at 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 951: 25 Tests
• SR101-Phe-CMK, 1 vial, #6151
• 10X Cellular Wash Buffer, 15 mL, #6164
• Fixative, 6 mL, #636
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 952: 100 Tests
• SR101-Phe-CMK, 4 vials, #6151
• 10X Cellular Wash Buffer, 60 mL, #6165
• Fixative, 6 mL, #636
• Hoechst 33342, 1 mL, #639
• Kit Manual