Far-Red Fluorescent FLICA® 660 Caspase-1 (YVAD) Assay Kit

1. Prepare samples and controls, 2. Dilute 10X Binding Buffer 1:10 with diH2O., 3. Wash cells in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer., 4. Dilute Annexin V-FITC 1:10 in PBS., 5. Dilute Propidium Iodide (PI) 1:2 in PBS., 6. Add diluted Annexin V-FITC to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells)., 7. Add diluted PI to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells)., 8. Incubate ~10 minutes, protected from light., 9. Dilute samples by adding 250 µL 1X Binding Buffer to each., 10. Analyze with a flow cytometer. Annexin V-FITC optimally excites at 494 nm and emits at 519 nm (typically read in FL1). PI optimally excites at 536 nm and has a peak emission at 617 nm (typically read in FL3).

Our FLICA® probes are cell permeant noncytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind with active caspase enzymes. We have developed a far-red excitation and emission spectra FLICA® 660 probe for the detection of cells bearing active caspase-1. As a part of the pathway to process inflammatory precursors, activation of caspase-1 represents one of many roles caspases have in cellular function and differentiation outside of apoptosis. Exposure of inflammatory effector cells like monocytes and macrophages to pathogen-associated molecular patterns (PAMPS), such as viral or bacterial DNA or RNA and bacterial cell wall components like LPS, will typically trigger conformational changes in NACHT leucine-rich repeat protein family (NLRP) proteins. This leads to oligomerization and assembly of a high molecular weight (~700 kDa) multimeric inflammasome complex, which leads to the conversion of pro-caspase-1 into the catalytically active form. Caspase-1, or interleukin-converting enzyme (ICE), proteolytically converts the proforms of interleukin 1ß (IL-1ß) and IL-18 in monocytes and macrophages. Please note that macrophages and monocytes have been shown to rapidly secrete caspase-1 upon activation. Activated caspase enzymes cleave proteins by recognizing a 3 or 4 amino acid sequence that must include an aspartic acid (D) residue in the P1 position. Our FLICA® 660 caspase-1 inhibitor probe contains the preferred binding sequence for caspase-1, Tyr-Val-Ala-Asp (YVAD). It should be noted that the YVAD binding sequence is also recognized by caspases 4 and 5. The YVAD binding sequence is labeled at the amino terminus end with a far-red fluorescent 660 dye and linked at the carboxyl end to a fluoromethyl ketone (FMK) reactive entity. The resulting cell permeant fluorescent molecule, 660-YVAD-FMK, optimally excites at 660 nm and emits between 685-690 nm. A conventional red HeNe laser with a 633 nm excitation provides excellent excitation efficiency, enabling cells labeled with FLICA® 660 to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities. Caspases, like most other crucial cell survival enzymes, are somewhat permissive in the target amino acid sequence they will recognize and cleave. Although FLICA® reagents contain the different amino acid target sequences preferred by each caspase, they can also recognize other active caspases when they are present. We encourage validation of caspase activity by an orthogonal technique. To use FLICA®, add it directly to the cell media, incubate, and wash. FLICA® is cell-permeant and will efficiently diffuse in and out of all cells. If there is an active caspase-1 enzyme inside the cell, it will covalently bind with FLICA 660-YVAD-FMK and retain the far-red fluorescent signal within the cell. The caspase does not cleave the covalently-bound FLICA®, and becomes inhibited from further enzymatic activity. Unbound FLICA® will diffuse out of the cell during the wash steps. Therefore, positive cells will retain a higher concentration of FLICA® and fluoresce brighter than negative cells. There is no interference from pro-caspases or inactive forms of the enzyme. After labeling with FLICA®, cells can be counter-stained with other reagents and fixed or frozen. FLICA® is for research use only. Not for use in diagnostic procedures.

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.