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ICT’s Serine Protease Kits:

• Are easy Just add the reagent directly to the cell culture and incubate.
• Are fast The reactions start within 15 minutes of addition to the cells, 
however we recommend an incubation of 1-4 hours. 
• Do not kill the cells The reagents may be incubated for several hours without killing the cells.
• Are brighter Carboxyfluorescein 
generates a brighter signal than FITC.
• Get into cells better Our reagents are nonpolar, so they pass right thru the cell membrane.
• Use whole living cells You can study apoptosis as it occurs in the cell. 
• No lysis or permeabilization steps required You don’t have to destroy the cell to study it. 
• Generate better data There is no interference from pro-enzymes or inactive forms of the enzyme.
• Do not use antibodies They use substrate and inhibitor peptide sequences linked to a fluorescent label.
• Are more reliable Only cells with active enzymes fluoresce. 
• Are flexible Protocols can be customized to fit your experiment. 
• Are qualitative and quantitative Read with a fluorescence plate reader, fluorescence microscope, or flow cytometer. 

Sample Protocol for FLISP™ Kits

1. Culture your cells individually or up to 1 x 10⁶ cells/mL. 
2. Induce apoptosis following your protocol. 
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the stock concentrate with 1X PBS to form the working solution. 
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling. 
6. Incubate 1-4 hours (or longer). 
7. Wash and spin cells twice, or let incubate for 1 hour with fresh media or 1x apoptosis wash buffer. 
8. If desired, label cells with Hoechst stain. 
9. If desired, label cells with Propidium Iodide. 
10. If desired, fix cells. 
11. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

Frequently Asked Questions

How sensitive is it? Our kits are very sensitive. They even detect background enzymes in the control cells.  Positive cells aften generate a signal 2-15x greater than the control population.
How long does it take? The reagents start to react within 15 minutes of addition to the cells, and can be incubated up to 72 hours without killing the cells. Typical incubation periods are 1-4 hours so you can start and finish your experiment in one day. The exact incubation time should be determined by your experimental protocol.  
What is “1 test”? As a general guide, “1 test” is a 300uL aliquot of your cell culture for testing on a fluorescence plate reader. Your cell culture may be quite different; the kits work with just a few cells for viewing under a fluorescence microscope, or up to 1x10⁶ cells/mL for flow cytometry.
How much reagent do I use? The reagents are provided as stabilized lyophilized powders. Just reconstitute the vial with DMSO, dilute with PBS, and then add a few uL of the diluted reagent to your cells. Your particular cell line may require more or less reagent. ICT’s kits can be customized by adapting the amount of reagent used and the length of the incubation periods. 
What wavelengths do I need?
The green cholinesterase, FLICA™, and FLISP™ reagents excite 
at 490nm and emit at 520nm. The red FLICA™ and FLISP™ reagents excite at 560nm and emit at 600nm. MR reagents excite at 540-590 and emit at >610nm. MitoPT™ excites at 488nm and emits at >590nm. Other filter pairings can be used which closely approximate these ranges. 
Will it work with fixed or paraffin cells? No, the FLISP™ kits will not work on fixed cells (but you can fix the cells after labeling), nor will they work with paraffin embedded cells. The reagents require the enzymes to be active, and fixation and paraffin inactivates the enzymes.
Will it work with frozen cells? The FLISP™ kits may work with some frozen cells and thin frozen sections. The reagents require the cell membrane to remain intact, and freezing often creates pores in the membranes, allowing the positive fluorescent signal to leave the cell. The Magic Red™ kits may work best with frozen cells when measuring total fluorescence with a plate reader.
How do I wash the cells?
The MitoPT™, FLICA™, FLISP™, and Cholinestease kits require a short wash step to remove any unbound background reagent. After incubating with the reagent, remove the media and spin with fresh media or ICT’s wash buffer. Adherent cells can be spun in a plate. If that is not feasible, or if your cells cannot withstand centrifugation, just replace the labeled media with fresh media or our wash buffer, and incubate 1 hour to allow any unbound reagent to diffuse back out of the cell. Remove this solution and analyze the cells. The MR kits do not require a wash step, so they may work best with sensitive cells, and frozen cells.