Immunochemistry Technologies, LLC

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#91 small FAM-FLICA Poly Caspases Apoptosis Detection Kit

   Assess neurodegeneration
   in vivo in live animal brain with FLIVO™
    or in vitro in cultured neurons with FLICA™
    or quantify cholinesterase in whole cells

Detect neurodegeneration in vivo with FLIVO™

Healthy rat brain (not green) neurons stained with Nissl (red).

Figure 1a: healthy rat brain neurons
fluoresce red. Read the paper (PDF).

Apoptotic rat neurons stained with FAM-FLIVO #981 (green) and Nissl (red).

Figure 1b: apoptotic rat neurons fluoresce green

Researchers can now quantify neuronal cell death in living animals using ICT’s new FLIVO™ fluorescent in vivo apoptosis detection kits. FLIVO™ is an injectable fluorescent probe that crosses the blood-brain barrier and is used to quantitate caspase activity in the brain. It is a direct stain; once labeled, tissues are ready for analysis and no further processing is necessary. FLIVO™ is very easy to use. Just inject it into the animal and let it circulate 30-45 minutes. Dying apoptotic neurons fluoresce green (Figure 2 or 3) or red (Figure 1), and can be counter-stained with other reagents (such as Nissl as seen in Figure 1).

Figure 1: Using FLIVO™ to monitor cell death, there is a clear distinction between healthy and apoptotic neurons. In this live animal brain study of diabetes, Dr. Thomas Morrow at the VA Hospital & University of Michigan was able to assess neurodegeneration in control (left) and 8-week STZ diabetic rats (right). 30 minutes prior to sacrifice, ICT’s green FAM-FLIVO™ in vivo apoptosis detection reagent (catalog #981) was injected intravenously to directly label dying caspase-positive apoptotic neurons within the live brain. After sacrifice, 20u frozen brain sections of the periaqueductal gray were prepared and counter-stained with red fluorescent Nissl to identify all neurons. Dying apoptotic neurons (right) exhibit dual staining with FAM-FLIVO™ (green) and Nissl (red). In this study, diabetic animals show greater levels of caspase activity in the brain (green, right) than control animals (not green, left). Data presented at the Society for Neuroscience 2006, poster #443.15/O10. Read a PDF of his paper

Figure 2: apoptotic neurons in sparrow brain

Figure 2: As part of his thesis work to examine naturally-occurring neuron death in seasonally-manipulated songbirds, Chris Thompson at the University of Washington used FAM-FLIVO (cat. # 981) to assess apoptosis in vivo. Mr. Thompson injected staurosporine (a protein kinase inhibitor that induces apoptosis) into the forebrain of a female house sparrow. ~20 hrs later, he injected FAM- FLIVO intravenously into the jugular. 30 min later, he sacrificed the bird via transcardial perfusion with heparinized saline and 4% paraformaldehyde. He postfixed the brain for 48 hrs, embedded it in gelatin, postfixed and cryoprotected the brain in 10% NBF and 20% sucrose for 48 hrs more. 40 um slices of the brain were made on a freezing microtome, and sections were mounted onto slides and coverslipped with ProLong antifade mountant. Apoptotic neurons with active caspases fluoresce green.

Deafferented bird brain is apoptotic in the NM (green) after labeling with FAM-FLIVO #981.

Figure 3a: control bird brain 3b: apoptotic bird neurons

Figure 3: In vivo labeling of caspase-positive neurons with FAM-FLIVO™ (green, catalog #981)) reveals a remarkable difference in the extent of apoptotic cell death in the nucleus magnocellularis (NM) of control (left) and deafferented (right) bird brains. Experimental procedure: remove the cochlea of 5-day-old chicks; survive for 6 hours; inject FAM-FLIVO™ into live animals 35 minutes prior to sacrifice; perfuse animals and fix the brain; vibrotome section the brain and mount. Cochlea was not removed in control animals. In the control bird (intact NM, left), a low level of background staining with FAM-FLIVO™ is presented uniformly in all cell bodies. In the experimental chick (right), nearly every auditory neuron in the deafferented NM is brightly stained with FAM-FLIVO™. FAM-FLIVO™ reveals that caspases are activated in every neuron of the NM just 6 hours after cochlear removal. Loss of stimulation from the cochlea, the auditory branch of the inner ear, induces a high level of apoptosis in the NM, second order auditory neurons in the chick brainstem. Data courtesy of Ms. Yuan Wang, University of Washington.

Grey-scale picture of rat brain ventricle.

Apoptotic subventricular cells fluoresce green after injection with FAM-FLIVO #981.

Figure 4a: grey image Figure 4b: apoptotic brain cells
fluoresce green

Figure 4: Apoptosis is revealed in subventricular cells of a rat brain after in vivo labeling with FAM-FLIVO™ (catalog #981, green, 4b). Caspase-positive cells fluoresce bright green. FAM-FLIVO™ was injected directly into the ventricle of a live rat brain (3a, red arrow points to the area in 4b), the animal was sacrificed and imaged. Data courtesy of Peggy Law, U of Toronto.

Detect neurodegeneration in vitro with FLICA™

ICT's FLICA™ kits make it easy to measure cell death and apoptosis in cultured neurons. Just add the reagent to the media, let it incubate 1- 4 hours, wash the cells and read with a fluorescence plate reader, microscope, or flow cytometer. Apoptotic cells fluoresce red or green.

Rat brain cortical cells become increasingly apoptotic with increased exposure to kainic acid. Caspase-positive cells (green) labeled with FAM-FLICA #92.
Figure 6: rat brain cortical cells

1 out of 2 oligodendrocytes is apoptotic (green) after staining with FAM-FLICA #92 and Hoechst (blue).

Figure 5: oligodendrocytes

Figure 5: This picture clearly distinguishes 1 apoptotic oligodendrocyte from many non-apoptotic cells: it fluoresces green after labeling with FAM-FLICA™. In this experiment, rat oligodendrocytes were grown on glass coverslips in Nunc 4-well plates and exposed to a drug that may or may not induce apoptosis. FAM-FLICA™ (green, catalog #92) was added in vitro to label caspase-positive cells. Hoechst (blue) was also added to label DNA of all cells. As only 1 of the oligodendrocytes had active caspases (green), this drug was not a potent inducer of apoptosis. Data courtesy of Brandon Miller, Ohio State University.

Figure 6. Apoptotic neurons can be easily distinguished from non-apoptotic cells using FAM-FLICA™ (kit #92). After exposure to hydrogen peroxide (H2O2)for 24 hours (upper left), 4 hours (upper right), and 40 minutes (lower left, FAM-FLICA™ was added to the cells for 1 hour to label caspase-positive cells green. Control cells (dark) were not exposed to H2O2 and exhibit little caspase activity (lower right). FAM-FLICA™ reveals that exposure to H2O2 induces apoptosis in rat brain cortical cells, and that continued exposure induces more caspases activity. Data courtesy of Babbin Tinner, QBM Cell Sciences.

2 neuroblastoma cells are apoptotic (green) after staining with FAM-FLICA #92 and Hoechst (blue).

Figure 7: neuroblastoma cells

Figure 7: FAM-FLICA™ reveals caspase activity in neuroblastoma cells after exposure to hydrogen peroxide (H2O2). In this experiment, 200,000 mouse N2a cells were seeded onto a coverslip in a 6-well plate and exposed to 50uM H2O2 for 20 hours. FAM-FLICA™ (green, catalog #92) was added to the media to label caspase-positive cells (green), and Hoechst was added to label DNA (blue). Apoptosis was induced in these 2 cells: they fluoresce green. Data courtesy of Joe Schowalter, University of Minnesota.

Labeling with FAM-FLICA (green) reveals that monocytes of Alzheimer's patients undergo a high level of apoptosis.
Fig. 8. Monocytes of Alzheimer’s disease (AD) patients (left pictures) become apoptotic and therefore cannot properly process amyloid beta. Control monocytes (right) differentiate into macrophages and efficiently phagocytize amyloid beta. Cells were exposed to amyloid beta (5 pg/ml) for 48 hours and then labeled with FAM-DEVD-FLICA™ (catalog #94) to stain caspase-3-positive cells green (bottom). In vitro staining with FLICA™ clearly distinguishes apoptotic AD monocytes (bottom left) from healthy control cells (bottom right). Data courtesy of Dr. Milan Fiala, UCLA. Read his press release of Oct 3, 2006.

Labeling with SR-FLICA (red, #932) reveals that monocytes of Alzheimer's patients undergo a high level of apoptosis (red and green, top).
Figure 9: Alzheimer's disease (top)
vs. normal human monocytes (bottom).

Figure 9: Alzheimer’s disease (AD) patients exhibit plaques of amyloid beta (AB) in the brain. In healthy people, amyloid beta is phagocytosed and cleared from the brain by macrophages. It is proposed that AD may be caused by a malfunction of the macrophage, which prevents them from properly clearing AB. This was tested by treating AD macrophages with curcumin, which is thought to enhance their function. Untreated control AD macrophages and curcumin-treated AD macrophages were exposed to FITC-labeled AB. It is seen that both groups engulf FITC-AB (they are green). The cells were then stained with ICT’s SR-DEVD Caspases 3&7 FLICA™ kit (catalog #932) to detect apoptosis: caspase-positive cells fluoresce red. In this experiment, essentially all of the untreated AD macrophages engulfed FITC-amyloid beta (green), but then became apoptotic (red). They are dually stained green and red. Curcumin-treated AD macrophages engulfed FITC-AB (green) and were not apoptotic, as evidenced by the lack of red fluorescence. It appears that in some AD patients, the macrophages engulf amyloid beta but undergo apoptosis before AB is cleared. Curcumin may protect macrophages from apoptosis; more studies are needed to validate this conclusion. Data courtesy of Dr. Milan Fiala, UCLA. Read his press release of Oct 3, 2006.

Figure 10: FAM-FLICA™was used to assess cell death in primay rat hippocampal neurons. Subjects were first-generation descendants of Sprague–Dawley albino rats. Hippocampi from PND 0, male pups were used for primary cultures of hippocampal neurons. Cells were plated on 25-mm poly-l-lysine-coated coverslips at 300,000 cells per coverslip. Cells were used at 4 or 8 days in vitro.

Composite (upper left)
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.

Figure 10: primary rat hippocampal neurons

Apoptosis (upper right)
3 Caspase-positive cells fluoresce green with FAM-FLICA™.

Necrosis (lower left)
2 Cells have permeabilized membranes and fluoresce red with PI.

All cells (lower right)
4 Cells are revealed by labeling DNA blue with Hoechst.

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

Quantify cholinesterase activity in vitro

With ICT’s new cholinesterase assay, researchers now have an easy way to directly quantitate and localize active cholinesterase enzymes in live, intact cells - no lysis or permeabilization steps are required.
This unique kit is not an ELISA and does not use antibodies - instead it is based on physostigmine, a known cholinesterase inhibitor, linked to a green fluorescent label, fluorescein (Fl). ICT’s Ph-Fl reagent is cell permeant, so you don’t have to lyse the cells or permeabilize the membranes. Because this kit does not use antibodies, there is no cross-reaction with inactive or pro forms of the enzyme. Only cells with the active cholinesterase enzyme will fluoresce.

Staining with Ph-F (#974) reveals cholinesterase activity in the nerve-muscle junction.

Figure 11. Localization of cholinesterase (catalog #974) in the nerve-muscle junctions (end-plates) in C57 mouse diaphragm muscle tissue. Arrows point to 4 of the 7 positive cells.

DAPI (blue left) labels all cells and can be used with ICT's reagents: Ph-F (center, green, #974) detects cholinesterase activity; SR-FLICA (red, right, #917) detects caspase activity.

Figure 12: cholinesterase and caspase activity in Jurkat cells.


	Figure 12. Varying expressions of DNA, cholinesterase, and caspase activity in apoptosis-induced Jurkat cells concurrently stained with DAPI, Ph-Fl (catalog #974), and SR-VAD-FMK (catalog #917). Cell 1 has no visible DNA (it is not blue), no caspase activity (it is not stained red), but it does exhibit active cholinesterase (it is green). Cell 2 reveals DNA, cholinesterase, and caspase activity at different intensities (it is blue, green, and red). Cell 3 has brightly stained DNA (it is blue), some cholinesterase activity (it is green), but no caspase activity (it is not red). Data courtesy of Dr. Zbigniew Darzynkiewicz of the Brander Cancer Center.

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800-829-3194 This page was updated on 04/04/2008 . Please contact us at 952-888-8788.