CASPASE APOPTOSIS PROTOCOLS FOR CELL DEATH DETECTION USING FLICA AND FLIVO FOR IN VITRO USE

Easily quantify cell death with ICT's fluorescent probes
Distinguish apoptosis from necrosis (using green or red probes)
Quantify cytotoxicity

Apoptosis vs. Necrosis with FAM-FLICA™ (green)

FAM-FLICA™(cat #92) was used to assess cell death in rat hippocampal neurons:
Composite
3 out of 4 cells are apoptotic (green). No cells were necrotic as both of the PI-positive cells were FLICA™-positive; they had compromised membranes and were probably in the late stages of apoptosis rather than necrosis.
Apoptosis
3 Caspase-positive cells fluoresce green with FAM-FLICA™.

Necrosis
2 Cells have permeabilized membranes and fluoresce red with PI.
All cells
4 Cells are revealed by labeling DNA blue with Hoechst.

Thank you! Data courtesy of Dr. Z. Kahraman Akozer, U of MD.

     ICT's Apoptosis vs. Necrosis FAM-FLICA™ kits (catalog #91 and 92) contain all the reagents you need to assess cell death in one easy protocol: use the FAM-FLICA™probe to label apoptotic cells green; Hoechst to label the DNA of all cells blue; and Propidium Iodide (PI) to label necrotic cells red. The kits also contain wash buffer and fixative.
     FAM-FLICA™ kits are not ELISAs and they do not use antibodies for detection. Instead, they use an inhibitor sequence of all caspases (VAD) linked to a green (carboxyfluorescein, FAM) fluorescent probe.  FLICA™= Fluorescent-Labeled Inhibitor of CAspases.  FLICA™ is cell-permeant so it automatically enters cells. If there is an active caspase enzyme inside the cell, it will bind to FLICA™ and retain the green fluorescent signal inside the cell. With FLICA™, only apoptotic cells will fluoresce green, so you can easily distinguish them from necrotic cells, which can be stained red with PI (included), or 7-AAD (sold separately). Hoechst is included to count cells with DNA (blue). Use ICT's red SR-FLICA™ kits to assess GFP-labeled cells; see below and the main FLICA™ page for more product information
     ICT's FLICA™ protocol can be easily integrated into most experiments and be completed in just a few hours: just add the reagents to your cells, incubate, wash, and read. Here is a detailed sample protocol using suspension cells: Read other FLICA™ protocols

1. Culture your cells up to 1 x 10⁶ cells/mL. 
2. Induce apoptosis following your protocol, and create positive and negative controls. 
3. Reconstitute FAM-FLICA™ with 50uL DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the FAM-FLICA™ stock concentrate with 200uL 1X PBS to form the working solution. 
5. Add ~10uL of the FAM-FLICA™ working solution directly to a 300-500uL aliquot of your cell culture for labeling. You may need to use more or less reagent than we suggest.
6. Incubate 1-4 hours. FAM-FLICA™ will start to react with the caspases within 15 minutes.
7. Spin cells ~5 minutes at 220g and carefully remove the FAM-FLICA™-labeled supernatant.
8. Add at least 400uL of the FLICA™ 1x apoptosis wash buffer (or use cell culture media).
9. Let the buffer incubate with the cells for 5-15 minutes protected from light (cells may be placed in the incubator).  
10. Carefully aspirate the buffer and discard.
11. Wash the cells up to 3 times by repeating Steps 7-10.  After the final wash, resuspend the cells in 1x apoptosis wash buffer for analysis. Some cells may need to be washed more than others depending on the type of instrument used.  Cells evaluated by flow cytometry may not need to be washed as much as cells evaluated in a plate reader or microscope, as the sheathing fluid acts as a wash buffer.  Cells analyzed in a plate reader often have to be washed the most, as the fluorometer measures total fluorescence within the well and any excess reagent will lead to higher RFUs. 
12. If desired, after the first wash, add 1.5uL Hoechst to label the DNA of cells blue.
13. If desired, after washing, add 1.5uL propidium iodide to label necrotic permeabilized cells red (or use red 7AAD, which is not included in FLICA™kits).
14. If desired, fix cells.
15. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

Order now!  1-800-829-3194.

FAM-FLICA™ (green) excites at 490 and emits at 520 nm.

Hoechst (blue) excites at 365 and emits at 480 nm.

Propidium iodide (red) excites at 490 and emits at 635 nm.

FAM-FLICA™ is compatible with DAPI, SR-FLICA™, Hoechst, PI, 7-AAD, and other fluorescent reagents for dual-staining studies.

Read the FAM manual

Read other FLICA protocols

References (pdf)

Order catalog #91 or 92 now!  1-800-829-3194.

Apoptosis vs. Necrosis with SR-FLICA™ (orange/red)

     ICT's SR-FLICA™ kits (catalog #916 and 917) enable you to quantitate cell death in one easy protocol: use the SR-FLICA™probe to label apoptotic cells orange/red; use Hoechst to label the DNA of all cells blue (included); and 7-AAD (catalog #6163, sold separately, and in ICT's Total Cytotoxicity Kits, see below) to label necrotic cells red. The kits also contain wash buffer and fixative.
     SR-FLICA™ kits are not ELISAs and they do not use antibodies for detection. Instead, they use an inhibitor sequence of caspases (VAD) linked to a reddish orange (sulforhodamine, SR) fluorescent probe.  FLICA™= Fluorescent-Labeled Inhibitor of CAspases.  FLICA™ is cell-permeant so it automatically enters cells. If there is an active caspase enzyme, it will bind to FLICA™ and retain the orange/red fluorescent signal inside the cell. With FLICA™, only apoptotic cells will fluoresce orange/red, so you can easily distinguish them from necrotic cells when stained red with 7-AAD (sold separately). Hoechst is included to count cells with DNA (blue). SR-FLICA™ kits work well to assess GFP-labeled cells; see the main FLICA™ page for more product information

In this experiment, K562 cells are labeled with SR-FLICA™ and 7AAD and analzyed via flow cytometry. Using SR-FLICA™, you can clearly identify 4 populations of cells: live, unstained cells; early apoptotic cells, which are only stained with SR-FLICA™; late apoptotic cells, which are stained with both SR-FLICA™ and 7AAD; and necrotic cells, which are only stained with 7AAD. 

  ICT's FLICA™ protocol can be easily integrated into most experiments and be completed in just a few hours: just add the reagents to your cells, incubate, wash, and read. Here is a detailed sample protocol using suspension cells: Read other FLICA protocols

1. Culture your cells up to 1 x 10⁶ cells/mL. 
2. Induce apoptosis following your protocol, and create positive and negative controls. 
3. Reconstitute SR-FLICA™ with 50uL DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the SR-FLICA™ stock concentrate with 200uL 1X PBS to form the working solution. 
5. Add ~10uL of the SR-FLICA™ working solution directly to a 300-500uL aliquot of your cell culture for labeling. You may need to use more or less reagent than we suggest.
6. Incubate 1-4 hours. SR-FLICA™ will start to react with the caspases within 15 minutes.
7. Spin cells ~5 minutes at 220g and carefully remove the SR-FLICA™-labeled supernatant.
8. Add at least 400uL of the FLICA™ 1x apoptosis wash buffer (or use cell culture media).
9. Let the buffer incubate with the cells for 5-15 minutes protected from light (cells may be placed in the incubator).  
10. Carefully aspirate the buffer and discard.
11. Wash the cells up to 3 times by repeating Steps 7-10.  After the final wash, resuspend the cells in 1x apoptosis wash buffer for analysis. Some cells may need to be washed more than others depending on the type of instrument used.  Cells evaluated by flow cytometry may not need to be washed as much as cells evaluated in a plate reader or microscope, as the sheathing fluid acts as a wash buffer.  Cells analyzed in a plate reader often have to be washed the most, as the fluorometer measures total fluorescence within the well and any excess reagent will lead to higher RFUs. 
12. If desired, after the first wash, add 1.5uL Hoechst to label the DNA of cells blue.
13. If desired, after washing, add 7AAD to label necrotic permeabilized cells red (sold separately).
14. If desired, fix cells.
15. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

Order now!  1-800-829-3194.

Thank you! FACS Data courtesy of Dr. Michael Olin, University of Minnesota.

SR-FLICA™ (orange/red) excites at 550 and emits at 590 nm.

Hoechst (blue) excites at 365 and emits at 480 nm.

7-AAD (red)  emits at 647 nm.

SR-FLICA™ is compatible with DAPI, FAM-FLICA™, Hoechst, 7-AAD, and other fluorescent reagents for dual-staining studies.

Read the red SR manual

Read other FLICA protocols

References (pdf)

Order catalog #916 or 917 now! Include 7AAD #6163 to label necrotic cells.  1-800-829-3194.

Total Cytotoxicity (red and green fluorescence)

Quantify 4 populations of cells: 
live; early apoptotic; late apoptotic, and necrotic.

Kit includes: CFSE, a green membrane stain; SR-FLICA™, an orangey-red apoptosis stain; and 7AAD, a red necrosis stain.

Read the TCT manual

Order catalog #971 or 972 now!  1-800-829-3194.

Please contact Sally Hed with questions 952-888-8788 x10 sally@immunochemistry.com .