Product Details
Catalog Number: KF17362
Applications: Apoptosis Detection
Type: Kit
Storage: Store at 2°C-8°C.
Shipping: Refrigerated (Polar Packs)
Format B: lyophilized
Species Reactivity: All
Product Sizes
SizeList PricePriceCart
25-50 Tests$229.00Add to Cart
Forget the lysates. Assay for apoptosis via caspase activity in whole, living cells with the FLICA® 660 Polycaspase Assay Kit:
  • Ex: 660 nm / Em: 680-690 nm
  • Whole cell analysis via flow cytometry or fluorescence microscopy
  • Flexible multiplexing with additional dyes or probes
  • Compatibility with GFP and green autofluorescence
  • Benchtop flow compatible

This in vitro caspase assay employs a new far-red fluorescent caspase inhibitor probe, FLICA® 660-VAD-FMK, to label active caspase enzymes in living cells or tissue samples. Image the fluorescent signal using fluorescence microscopy or analyze samples for caspase activity by flow cytometry. The cell permeant FLICA caspase detection probe, 660-VAD-FMK, is comprised of an affinity inhibitor peptide sequence (VAD) and a fluoromethyl ketone (FMK) moiety that enable an irreversible, covalent binding event with active caspase enzymes. This newest FLICA probe is labeled with a far-red fluorescent 660 dye reporter, enabling detection via common fluorescence detection methods.

Protocol
FLICA™ (Fluorescent-Labeled Inhibitor of Caspases) assays offer a simple yet accurate method to measure individual or pan-caspase activity in whole cells. Two sample protocols are outlined below. Detailed instructions are supplied with the kit.

Suspension Cells

  1. Culture your cells to a concentration of 2-5 x 105 cells/mL.
  2. Prepare experimental and control populations.
  3. Reconstitute the reagent with 50µL DMSO to form the stock concentrate (may be frozen for future use).
  4. Dilute the stock concentrate with 200µL PBS to form the working solution.
  5. Add working solution directly to samples and controls at a ratio of 1:30 – 1:60. E.g., add 5-10 µL of working solution to a 300 uL aliquot of suspension cells. Flow cytometry requires less reagent; microscopy applications require more reagent per sample.
  6. Incubate 15 - 45 minutes. 
  7. Wash and spin cells two or three times.
  8. If desired, label cells with Hoechst stain, DAPI, or other compatible fluorescent markers.
  9. If desired, fix cells with fixative included in kits.
  10. Analyze data using a fluorescence microscope or flow cytometer.

Tissue Sections

  1. Prepare experimental and control populations.
  2. Prepare thin frozen tissue sections appropriate to the experiment. Allow sections to thaw before staining with FLICA reagent.
  3. Reconstitute each vial of FLICA with 50 uL DMSO to form the stock concentrate.
  4. Dilute FLICA stock concentrate 1:50 in PBS to form the tissue section staining solution (TSSS). Use TSSS within 15 minutes of preparation. 
  5. Add enough TSSS to cover the surface of the tissues and incubate 30 - 60 minutes protected from light. 
  6. Wash with TBSt, PBSt, or 1X Cellular Wash Buffer (twice for 5 min).
  7. Set slides in slide incubation dish containing 1X Cellular Wash Buffer.
  8. Stain nuclei with DAPI or Hoechst, and apply coverslip.
  9. Image with appropriate fluorescence microscopy filters.
  10. Store samples at 2-8°C for short-term storage; staining will last at -20° C for long periods.
Images

THP-1 cells were treated with either a placebo or PMA and LPS. After treatment and trypsinization, FLICA 660-YVAD-FMK far-red caspase-1 reagent was added to the culture media. Flow cytometric analysis of the positive control sample (top) reveals a slight increase in cells bearing active caspase-1 enzyme when compared to the population of placebo-treated cells (bottom). An 8:1 differential was achieved with the positive control treatment.

THP-1 cells were treated with either a placebo or PMA and LPS. After treatment and trypsinization, FLICA 660-YVAD-FMK far-red caspase-1 reagent was added to the culture media. Flow cytometric analysis of the positive control sample (top) reveals a slight increase in cells bearing active caspase-1 enzyme when compared to the population of placebo-treated cells (bottom). An 8:1 differential was achieved with the positive control treatment.