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Are the Cells Apoptotic or Necrotic?

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Find out how your cells are responding in vitro.
Easily quantify cell death with ICTs fluorescent probes. Distinguish apoptosis from necrosis (using green or red probes) and quantify cytotoxicity.

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


ICTs Apoptosis vs. Necrosis FAM-FLICA™ kits (catalog # ICT-91 and ICT-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. Hoechst is included to count cells with DNA (blue). Use ICTs red SR-FLICA™ kits to assess GFP-labeled cells.

Protocol


ICTs 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 here.)

1. Culture your cells up to 1 x 10^6 cells/mL.
2. Induce apoptosis following your protocol, and create positive and negative controls.
3. Reconstitute FAM-FLICA™ with 50 uL DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the FAM-FLICA™ stock concentrate with 200 uL 1X PBS to form the working solution.
5. Add ~10 uL of the FAM-FLICA™ working solution directly to a 300-500 uL 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 220 g 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 1.5 uL Hoechst to label the DNA of cells blue.
13. If desired, after washing, add 1.5 uL 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.

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


ICTs SR-FLICA™ kits (catalog # ICT-916 and ICT-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 (in ICTs 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.

Protocol


ICTs 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 here.)

1. Culture your cells up to 1 x 10^6 cells/mL.
2. Induce apoptosis following your protocol, and create positive and negative controls.
3. Reconstitute SR-FLICA™ with 50 uL DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the SR-FLICA™ stock concentrate with 200 uL 1X PBS to form the working solution.
5. Add ~10 uL 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 220 g and carefully remove the SR-FLICA™-labeled supernatant.
8. Add at least 400 uL 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.

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