Vybrant Apoptosis Assay Kit #1

Vitalità cellulare, proliferazione e funzionalità
cellulare
 La sopravvivenza cellulare, la proliferazione e altre
importanti funzioni vitali - incluso apoptosi, adesione
cellulare, chemiotassi, resistenza ad agenti farmacologici,
endocitosi, secrezione e trasduzione del segnale possono essere stimolate e monitorate con vari agenti
chimici e reagenti biologici. Molti di questi processi
inducono cambiamenti nella concentrazione di radicali
liberi, di ioni o del potenziale di membrana che possono
essere seguiti con indicatori fluorescenti appositi.
 Vedremo rapidamente alcuni kit attualmente in commercio
che permettono di valutare questi processi biologici. Ci
occuperemo in particolare di kit per:
1.
Saggi di proliferazione cellulare e sopravvivenza
2.
Saggi di apoptosi
3.
Saggi di adesione, chemiotassi e glutatione.
Esistono anche sonde specifiche per valutare
1.
Endocitosi ed esocitosi
2.
Recettori di neurotrasmettitori
3.
Canali ionici e trasportatori
1. Assays for Cell Viability, Cell
Proliferation and Cell Cycle
1.1 I saggi di sopravvivenza e citotossicità sono usati
prevalentemente per valutare la proporzione di cellule vive e
morte in una certa popolazione. Al contrario i saggi di
proliferazione sono usati prevalentemente per monitorare la
velocità di crescita di una popolazioe cellulare. Questi saggi
basati sulla fluorescenza sono in generale meno rischiosi e
meno costosi di tecniche radioisotopiche, più sensibili dei
metodi colorimetrici e più convenienti dei test su animali.
In particolare i test fluorometrici di sopravvivenza e
citotossicità sono facili da utilizzare con l’uso di un
microscopio a fluorescenza, di un fluorimetro, di un
lettore di micropiastra o di un citofluorimetro. Vedremo
adesso reagenti appositamente studiati per saggi di
sopravvivenza e citotossictà in una ampio spettro di
cellule, incluse quelle di origine animale come pure
lieviti e batteri. Per esempio vediamo il seguente kit:
LIVE/DEAD Viability/Cytotoxicity Kit
(sopravvivenza)
Principle of the Method (Molecular Probes)
Il kit LIVE/DEAD® Viability/Cytotoxicity fornisce un test a due
colori che è basato sulla determinazione simultanea delle
cellule vive e di quelle morte utilizzando due sonde che
misurano due parametri particolari:
sopravvivenza — attività delle esterasi intracellulari e integrità
della membrana cellulare
Questo kit è anche usato per quantificare la morte cellulare
per apoptosi e la citotossicità cellula-mediata
Le cellule vive si riconoscono per la presenza ubiquitaria
di attività esterasica. Questa si valuta per conversione
enzimatica di calceina AM (substrato delle esterasi) non
fluorescente e permeabile attraverso la membrana in
calceina fluorescente. Questa forma è ritenuta all’interno
delle cellule vive che si colorano in verde. EthD-1 (etidio
omodimero 1) entra invece nelle cellule con membrana
danneggiata, si lega agli acidi nucleici e produce una luce
rossa brillante (cellule morte). EthD-1 non pasa attraverso
membrane cellulari sane.
Live and dead kangaroo rat (PtK2) cells stained with ethidium
homodimer-1 and the esterase substrate calcein AM
A mixed population of live
and isopropyl alcohol–killed
Micrococcus luteus and
Bacillus cereus stained with
the LIVE/DEAD BacLight
Bacterial Viability Kit.
Bacteria with intact cell
membranes exhibit green
fluorescence, whereas
bacteria with damaged
membranes exhibit red
fluorescence.
La proliferazione cellulare e la caratterizzazione di
sostanze in grado di promuovere o ritardare la
proliferazione sono aree interessanti di studio per la
biologia cellulare e la messa a punto di nuovi farmaci. Per
vedere semplicemente le cellule e numerarle, le colorazioni
fluorescenti che identificano le cellule in base a
caratteristiche morfologiche, possono essere sufficienti. La
particolare sensibilità di alcuni kit fluorescenti permette
anche la quantificazione di alcuni virus.
Qualora si voglia invece valutare la proliferazione cellulare,
di quantità
di DNA
la**La
cosavariazione
diviene più
difficoltosa
perché, attualmente, non vi
*BrdU
è incorporato nelle cellule
è
associabile
alle
diverse
fasi
del
sono dye fluorescenti che possano essere incamerati nella
nuove e viene riconosciuto grazie
ciclo cellulare.
è marcato
cellula
duranteIlilDNA
processo
mitotico . Per questo motivo la
adpoi
unse
anticorpo che evidenzia
con un fluoroforo
maggior
parte deispecifico
saggi di eproliferazione
stimano il numero
quindi le cellule che proliferano.
ne
valuta
la
quantità.
di cellule per incorporazione di un marcatore triziato,
bromo deossi uridina (BrdU, analogo della timidina)* nelle
cellule, oppure misurando il contenuto totale di acidi
nucleici** o di proteine nel lisato cellulare.
Human lymphoma cells were allowed to proliferate and then stained
using the ABSOLUTE-S SBIP Cell Proliferation Assay Kit. Proliferating
cells that have incorporated 5-bromo-2'-deoxyuridine (BrdU) stain
green, while necrotic cells stain red.
Detection of cell proliferation by BrdU incorporation. 3T3 cells were
pulsed with BrdU for 30 minutes before fixation. BrdU incorporated into
the DNA of proliferating cells was detected with an anti-BrdU antibody
that was labeled with the Zenon Alexa Fluor 488 Mouse IgG1 Labeling
Kit.
1.3 Progression of the cell cycle
through G1-phase and into Sphase is controlled in part by a
series of serine/threonine kinase
complexes, consisting of a
cyclin regulatory subunit and
catalytic subunit referred to as a
cyclin-dependent kinase (cdk).
Binding of the cyclin to the cdk
activates the complex, which
promotes cell-cycle progression
by phosphorylation of specific
target proteins.
The different phases of the cell
cycle. In the first phase (G1) the
cell grows. When it has reached
a certain size it enters the phase
of DNA-synthesis (S) where the
chromosomes are duplicated.
During the next phase (G2) the
cell prepares itself for division.
During mitosis (M) the
chromosomes are separated and
segregated to the daughter
cells, which thereby get exactly
the same chromosome set up.
The cells are then back in G1
and the cell cycle is completed.
At least five proteins have been identified as G1-phase
cyclins (C, D1, D2, D3, E), of which the three D cyclins form
a closely related group. Cyclin C associates with cdk8,
while cyclin E activates cdk2. The D1 and D2 cyclins
associate with both cdk4 and cdk6; activity of these
complexes has been detected as early as mid-G1 phase.
Cyclin D3 can also activate cdk4 and cdk6, but D3associated cdk activity is found only at the G1/S transition.
Ciclina C
Ciclina D1
Ciclina E
cdk8
cdk4, cdk6
mid G1
cdk2
Ciclina D2
cdk4, cdk6
mid G1
Ciclina D3
cdk4, cdk6
ma solo nella transizione G1/S
The D cyclins, in particular, play an important role in
regulatory decisions controlling the progression of the cell
cycle; overexpression of these regulatory proteins is
associated with a wide variety of proliferative diseases
including breast and gastric cancers. For the detection of
these important cell-cycle control proteins, Molecular
Probes offers a monoclonal antibody against each
individual D cyclin — mouse IgG2a monoclonal DCS-6
anti–cyclin D1 (A21320), mouse IgG2b monoclonal DCS5.2 anti–cyclin D2 (A21321) and mouse IgG1 monoclonal
DCS-22 anti–cyclin D3 (A21322).
2. Apoptosis assay
What is apoptosis?
There are two ways in which cells die:
they are killed by injurious agents.
they are induced to commit suicide
Cells that are damaged by injury, such as by mechanical
damage or exposure to toxic chemicals undergo a
characteristic series of changes:
 they (and their organelles like mitochondria) swell
(because the ability of the plasma membrane to
control the passage of ions and water is disrupted).
 the cell contents leak out, leading to inflammation of
surrounding tissues.
Cells that are induced to commit suicide:

Shrink and develop bubble-like blebs on their
surface

Have the chromatin (DNA and protein) in their
nucleus degraded

Their mitochondria break down with the release of
cytochrome c

Break into small, membrane-wrapped, fragments

Phosphatidylserine, which is normally hidden within
the plasma membrane, is exposed on the surface

Phosphatidylserine is bound by receptors on
phagocytic cells like macrophages and dendritic
cells which then engulf the cell fragments

The phagocytic cells secrete cytokines that inhibit
inflammation
The pattern of events in death by suicide is so orderly that
the process is often called programmed cell death or
PCD. The cellular machinery of programmed cell death
turns out to be as intrinsic to the cell as, say, MITOSIS.
Programmed cell death is also called apoptosis.
Why should a cell commit suicide?
There are two different reasons.
1. Programmed cell death is as needed for proper
development as mitosis is.
Examples:
The formation of the fingers and toes of the fetus requires
the removal, by apoptosis, of the tissue between them.
The sloughing off of the inner lining of the uterus (the
endometrium) at the start of menstruation occurs by
apoptosis.
The formation of the proper connections (synapses)
between neurons in the brain requires that surplus cells be
eliminated by apoptosis.
2. Programmed cell death is needed to destroy cells that
represent a threat to the integrity of the organism.
Examples

Cells infected with viruses (cytotoxic T lymphocytes kill
virus-infected cells by inducing apoptosis)

Cells of the immune system (as cell-mediated immune
responses wane, the effector cells must be removed to
prevent them from attacking body constituents)

Cells with DNA damage (cells respond to DNA
damage by increasing their production of p53 (p53 is a
potent inducer of apoptosis)

Cancer cells
As with cell viability, no single parameter fully defines cell
death in all systems; therefore, it is often advantageous to
use several different approaches when studying apoptosis.
Several methods have been developed to distinguish live
cells from early and late apoptotic cells and from necrotic
cells.
Apoptotic cells are typically eliminated by phagocytosis;
thus, apoptotic cells that have been selectively labeled with
a fluorescent dye can potentially be used as tracers for
phagocytosis.
Several kits have been developed to assay and detect
apoptotic cells, based on the different molecular processes
involved in the mechanism. The following is a list
concerning these different ready-kits:
2.1 DNA Stains for Detecting Apoptotic Cells
The characteristic breakdown of the nucleus during
apoptosis comprises collapse and fragmentation of the
chromatin, degradation of the nuclear envelope and nuclear
blebbing, resulting in the formation of micronuclei. Nucleic
acid stains can be useful tools for identifying even low
numbers of apoptotic cells in cell populations.
2.2 Vybrant Apoptosis Assay Kit
Vybrant Apoptosis Assay Kit #4 detects apoptosis based on
changes that occur in the permeability of cell membranes.
This kit contains ready-to-use solutions of both the YOPRO-1 and propidium iodide nucleic acid stains. YO-PRO1 nucleic acid stain selectively passes through the plasma
membranes of apoptotic cells and labels them with
moderate green fluorescence. Necrotic cells are stained
with the red-fluorescent propidium iodide, a DNA-selective
dye that is membrane impermeant but that easily passes
through the compromised plasma membranes of necrotic
cells.
2.3. Vybrant Apoptosis Assay Kit #1
With the Vybrant Apoptosis Assay Kit #1 (V13240),
apoptotic cells are detected based on the externalization of
phosphatidylserine.
2.4. Apoptosis Assays Based on Protease Activity
A distinctive feature of the early stages of apoptosis is the
activation of caspase enzymes, the name applied to
cysteine–aspartic acid specific proteases. Caspases are a
key component of the apoptotic machinery of cells,
participating in an enzyme cascade that results in cellular
disassembly. The Vybrant FAM Caspase Assay Kits employ
a novel approach for caspase detection that is based on a
fluorescent caspase inhibitor (FLICA methodology).
2.5. Mitochondrial Stains for Detecting Apoptosis
The green-fluorescent dye JC-1 exists as a monomer at low
concentrations or at low membrane potential. However, at
higher concentrations or at higher membrane potentials, JC-1
forms red-fluorescent "J-aggregates" that exhibit a broad
excitation spectrum and an emission maximum at ~590 nm.
Thus, the emission of this cyanine dye has been widely used
to follow the changes in mitochondrial membrane potential
that occur as a result of apoptosis.
Rhodamine 123 is a cell-permeant, cationic, fluorescent
dye that is readily sequestered by active mitochondria
without inducing cytotoxic effects. Uptake and equilibration
of rhodamine 123 is rapid — a few minutes — as compared
with other membrane potential–sensitive dyes, which may
take 30 minutes or longer. Although not aldehyde-fixable,
rhodamine 123 allows for quick and easy detection of
apoptotic cells.
Cultured human pre-adipocytes loaded with the ratiometric
mitochondrial potential indicator JC-1 (T3168) at 5 µM for 30 minutes
at 37°C. In live cells, JC-1 exists either as a green-fluorescent
monomer at depolarized membrane potentials or as an orangefluorescent J-aggregate at hyperpolarized membrane potentials.
3.1 Cell Adhesion
The fundamental role of cell–cell and cell–matrix adhesion in the
morphology and development of organisms, organs and tissues has
made identification of molecular mediators of cell adhesion an
important research focus in cell biology. In a typical fluorescencebased cell adhesion assay, unlabeled cell monolayers in multiwell
plates are incubated with fluorescently labeled cells and then
washed to separate the adherent and nonadherent populations. Cell
adhesion can then be determined simply by correlating the retained
fluorescence with cell number.
An ideal fluorescent marker will retain proportionality between
fluorescence and cell number and introduce minimal interference
with the cell adhesion process. Because adhesion is a cell-
surface phenomenon, cytoplasmic markers that can be passively
loaded are preferable to compounds that label cell-surface
molecules, provided they are retained in the cell for the duration
of the experiment or their leakage rate can be independently
measured. Adhesion of fluorescent dye–labeled cells to matrices
such as bone an be directly observed by fluorescence
microscopy using cells loaded with the permeant live-cell tracers.
3.1 Vybrant Cell Adhesion Assay Kit
The Vybrant Cell Adhesion Assay Kit utilizes calcein AM to
provide a fast and sensitive method for measuring cell–cell
or cell–substratum adhesion. Calcein AM is nonfluorescent
but, once loaded into cells, is cleaved by endogenous
esterases to produce calcein, a highly fluorescent and wellretained dye. Calcein provides a bright green-fluorescent,
pH-independent, cytoplasmic cell marker that does not
appear to affect the cell adhesion process.
3.2 Fluorescent Gelatin and Type IV Collagen
Collagen is a major component of the extracellular matrix
and, in vertebrates, constitutes approximately 25% of total
cellular protein. This important protein not only serves a
structural role, but also is important in cell adhesion and
migration. Specific collagen receptors, fibronectin and a
number of other proteins involved in cell–cell and cell–
surface adhesion have been demonstrated to bind collagen
and gelatin (denatured collagen). Molecular Probes
prepares fluorescent conjugates of gelatin and type IV
collagen the principal collagen in basement membranes.
3.3 Anti-Fibronectin Antibody
Fibronectin is a large glycoprotein that is found in both
plasma and in the extracellular matrix. The protein is coded
by a single gene, but alternate RNA splicing gives rise to
several fibronectin isoforms that play important roles in
cellular adhesion and migration, blood clotting and
phagocytosis. The apparent function of fibronectin is to
mediate cell attachment via interactions with both cell-
surface receptors and components of the extracellular
matrix such as heparin, fibrinogen and collagen.
Molecular Probes offers a chicken IgY anti-fibronectin
(human) antibody (A21316, Anti-Fibronectin (Human)
Chicken IgY Fraction) for detecting fibronectin in Western
blotting and immunohistochemistry applications, as well as
several fluorescent anti–chicken IgG secondary antibodies
that recognize this antibody.
3.2 Chemotaxis Assays
Chemotaxis, defined as directed cell motion toward an
extracellular gradient, plays an important role during
fertilization, inflammation, wound healing and
hematopoiesis. Measurement of chemotaxis usually has
relied on determination of the number of viable cells that
have migrated through a special "chemotaxis chamber." The
probes used to follow chemotaxis in live cells typically are
the same esterase substrates that are useful for assaying
cell viability and cell adhesion.
The primary esterase substrates used for this purpose are
calcein AM (C1430, C3099, C3100MP), BCECF AM
(B1150, B1170, B3051) and CellTracker Green CMFDA
(C2925, C7025). Calcein AM does not interfere with
lymphocyte proliferation or with granulocyte or neutrophil
chemotaxis or superoxide production, and, unlike BCECF
AM, calcein AM does not affect chemotaxis in leukocytes.
3.3 Glutathione Determination
The tripeptide glutathione ( -L-glutamyl-L-cysteinylglycine)
is the most abundant (up to 10 mM in the cytoplasm and
about 1 mM in blood) and important non-protein thiol in
mammalian cells. Glutathione plays a central role in
protecting cells of all organs, including the brain, against
damage produced by free radicals, oxidants and
electrophiles. Several fluorescent reagents have been
proposed for determining cellular levels of glutathione and
glutathione S-transferase (GST), which catalyzes the
formation of glutathione S-conjugates, but no probe is
without drawbacks in quantitative studies of live cells.
The high but variable levels of intracellular glutathione and
the multitude of GST isozymes make kinetic measurements
under saturating substrate conditions difficult or impossible.
Isozymes of GST vary both in abundance and activity,
further complicating the analysis. Moreover, the
fluorescent reagents designed to measure glutathione may
react with intracellular thiols other than glutathione,
including proteins in glutathione-depleted cells.
Therefore, precautions must be taken in applying the
reagents mentioned here to quantitate either glutathione or
GST in cells. A useful strategy is to test a variety of
glutathione-sensitive dyes — those requiring glutathione Stransferase activity, as well as GST-independent
fluorophores — under controlled experimental conditions in
which glutathione is depleted.
3.3.1 Glutathione Determination with Monochlorobimane
Cell-permeant monochlorobimane (mBCl, M1381MP),
which is essentially nonfluorescent until conjugated to
thiols, has long been the preferred thiol-reactive probe for
quantitating glutathione levels in cells and for measuring
GST activity. Because the blue-fluorescent glutathione
adduct of monochlorobimane eventually accumulates in the
nucleus, it is not a reliable indicator of the nuclear and
cytoplasmic distribution of cellular glutathione. Tissue
glutathione levels can also be measured fluorometrically by
adding both monochlorobimane and glutathione Stransferase to tissue homogenates
3.3.2 Glutathione Determination with Visible Light–
Excitable Thiol-Reactive Probes
CellTracker Green CMFDA (5-chloromethylfluorescein
diacetate, C2925, C7025) is a useful alternative to the UV
light–excitable monochlorobimane for determining levels of
intracellular glutathione. CellTracker Green CMFDA can be
excited by the argon-ion laser. Like CMFDA, chloromethyl
SNARF-1 acetate (C6826) forms adducts with intracellular
thiols that are well retained by viable cell.
Human neutrophils loaded with 5-(and-6)-chloromethyl SNARF-1 acetate
Probes for Neurotransmitter Receptors
Because receptor-mediated signal transduction underlies
much of what occurs in cellular biochemistry and physiology,
fluorescent receptor ligands can provide a sensitive means
of identifying and localizing some of the most pivotal
molecules in cell biology. Molecular Probes offers
fluorescently labeled and unlabeled ligands for various
cellular receptors, ion channels and ion carriers. Many of
these site-selective fluorescent probes may be used on live
or fixed cells, as well as in cell-free extracts.
In particular, we would like to highlight those ligands
conjugated to the green-fluorescent Alexa Fluor 488,
BODIPY FL and Oregon Green 514 dyes and the redfluorescent Alexa Fluor 594 and Texas Red dyes, which
provide extremely bright signals and superior photostability.
The high sensitivity and selectivity of these fluorescent
probes make them especially good candidates for
measuring low-abundance receptors
Un esempio: α-Bungarotoxin Probes for
Nicotinic Acetylcholine Receptors
Nicotinic acetylcholine receptors (nicotinic AChRs) are
neurotransmitter-gated ion channels that produce an
increase in Na+ and K+ permeability, depolarization and
excitation upon activation by acetylcholine. α -Bungarotoxin,
a 74–amino acid peptide extracted from Bungarus
multicinctus venom, binds with high affinity to the α -subunit
of the nicotinic AChR of neuromuscular junctions.
Molecular Probes provides an extensive selection of
fluorescent α -bungarotoxin conjugates (Table 16.4, alphaBungarotoxin and Conjugates) to facilitate visualization of
nicotinic AChRs with a variety of instrumentation. We attach
approximately one fluorophore to each molecule of α bungarotoxin, thus retaining optimal binding specificity. The
labeled bungarotoxins are then chromatographically
separated from unlabeled molecules to ensure adequate
labeling of the product.
Fluorescent α-bungarotoxins have been used in a variety of
informative investigations of the nicotinic AChR to:
• Correlate receptor clustering during neuromuscular
development with tyrosine phosphorylation of the
receptor.
• Document nicotinic AChR cluster formation after
myoblast fusion.
• Quantitate nicotinic AChRs in a study that showed that
several isotypes of agrin, a component of synaptic basal
lamina, help trigger the nicotinic AChR aggregation that
occurs during neuromuscular junction formation.
• Detect reinnervation of adult muscle after nerve damage
and to identify and visualize endplates.
Amplex Red Acetylcholine/Acetylcholinesterase
Assay Kit
The action of acetylcholine (ACh) at neuromuscular
junctions is regulated by acetylcholinesterase (AChE), the
enzyme that hydrolyzes ACh to choline and acetate. The
Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit
provides an ultrasensitive method for continuously
monitoring AChE activity and for detecting Ach. Other
potential uses for this kit include screening for AChE
inhibitors and measuring the release of ACh from
synaptosomes.
AChE activity is monitored indirectly using the Amplex Red
reagent, a highly sensitive and stable fluorogenic probe for
H2O2 that is useful in numerous assays for enzymes and
other analytes. First, AChE converts the acetylcholine
substrate to choline. Choline is in turn oxidized by choline
oxidase to betaine and H2O2, the latter of which, in the
presence of horseradish peroxidase, reacts with the
Amplex Red reagent to generate the red-fluorescent
product resorufin with excitation/emission maxima of
~570/585 nm
(HRP)
Per altri neurotrasmettitori
Pirenzepine Probes for Muscarinic Acetylcholine Receptors:
Unlike nicotinic AChRs, muscarinic acetylcholine receptors
(muscarinic AChRs) are G-protein–coupled receptors that
produce either excitatory or inhibitory responses and are not
necessarily associated with changes in ion permeability.
Fluorescent derivatives of pirenzepine are selective antagonists
for the M1 muscarinic AChR. The green-fluorescent BODIPY FL
and red-fluorescent BODIPY 558/568 derivatives retain
pirenzepine's specificity for the M1 muscarinic receptor and
exhibit similar inhibition and displacement profiles.
Prazosin Probes for α1-Adrenergic Receptors: the greenfluorescent BODIPY FL and red-fluorescent BODIPY 558/568
derivatives of prazosin, a high-affinity antagonist for the α1adrenergic receptor are available. These derivatives can be used
to localize the α1-adrenergic receptors on cultured cortical
neurons.
BODIPY TMR-X Muscimol for the GABAA Receptor: Muscimol is
a powerful agonist of the GABAA receptor and has been widely
used to reversibly inactivate localized groups of neurons. With
the introduction of the BODIPY TMR-X muscimol conjugate
researchers can avoid using radioactive methods to map the
distribution of the drug in the central nervous system, as well as
to detect the presence of GABAA receptors on cell surfaces.
Neuropeptide Probes for the Neurokinin Receptors: Substance P is
an agonist of the neurokinin 1 (NK1) receptor, a member of the
seven-domain transmembrane GPCRs. Substance P is a tachykinin
— one of a group of neuropeptides that are primarily involved in the
mediation of inflammatory responses. NK1 receptor probes include:
• Alexa Fluor 488 and Oregon Green 488 conjugates of
substance P: the Alexa Fluor 488 and Oregon Green 488 dyes
virtually match fluorescein's excitation and emission spectra
but have the additional benefits of superior photostability and
lower pH sensitivity.
• Fluorescein conjugate of substance P
• Tetramethylrhodamine conjugate of substance P : substance P
labeled with the photostable, orange-fluorescent
tetramethylrhodamine dye offers researchers another option for
multicolor experiments.
Anti–NMDA Receptor Antibodies: NMDA receptors constitute
cation channels of the CNS that are gated by the excitatory
neurotransmitter L-glutamate. Activation of NMDA receptors is
essential for inducing LTP, a form of activity-dependent synaptic
plasticity that is implicated in the learning process in animal
behavioral models. The biophysical properties of NMDA
receptor channels contributing to LTP include Ca2+ permeability,
voltage-dependent Mg2+ blocking and slow-gating kinetics.
NMDA receptor–channel activities play a role in neuronal
development and in disorders such as epilepsy and ischemic
neuronal cell death.
In vitro reconstitution experiments with the cloned NMDA
receptor subunit 1 and any one of the four NMDA receptor
subunits 2A, 2B, 2C and 2D revealed that the physical properties
of the heteromeric NMDA receptor channel appear to be
imparted by the particular NMDA receptor subunit 2. NMDA
receptor subunits 2A and 2B are detected predominantly in the
hippocampus and cortex, whereas 2C is found mainly in the
cerebellum. Thus, cellular expression profiles of the NMDA
receptor subunits 2A, 2B, 2C and 2D may contribute to the
biophysical properties of NMDA receptors in specific central
neurons.
NMDA receptor overview
 NMDA receptors are composed of assemblies of NR1 subunits
and NR2 subunits, which can be one of four separate gene
products (NR2A-D). Expression of both subunits are required to
form functional channels. The glutamate binding domain is
formed at the junction of NR1 and NR2 subunits (hence the
need for both subunits to be expressed). In addition to
glutamate, the NMDA receptor requires a co-agonist, glycine, to
bind to allow the receptor to function. The glycine binding site is
found on the NR1 subunit. The NR2B subunit also has a
binding site for ployamines, regulatory molecules that modulate
the functioning of the NMDA receptor.
There are affinity-purified rabbit polyclonal antibodies to NMDA
receptor subunits 2A, 2B and 2C. The anti–NMDA receptor
subunit 2A and 2B antibodies were generated against fusion
proteins containing amino acid residues 1253–1391 of subunit 2A
and 984–1104 of subunit 2B, respectively. These two antibodies
are active against mouse, rat and human forms of the antigens
and are specific for the subunit against which they were
generated. In contrast, the anti–NMDA receptor subunit 2C
antibody was generated against amino acid residues 25–130 of
subunit 2C and recognizes the 140,000-dalton subunit 2C, as well
as the 180,000-dalton subunit 2A and subunit 2B from mouse, rat
and human. These three affinity-purified antibodies are suitable
for immunohistochemistry, Western blots, enzyme-linked
immunosorbent assays (ELISAs) and immunoprecipitations.
Probes for
Ion Channels and Carriers
Probes for Ca2+ Channels and Carriers
In both excitable and nonexcitable cells, intracellular Ca2+ levels
modulate a multitude of vital cellular processes — including
gene expression, cell viability, cell proliferation, cell motility and
cell shape and volume regulation — thereby playing a key role
in regulating cell responses to external activating agents. These
dynamic changes in intracellular Ca2+ levels are regulated by
ligand-gated and G-protein–coupled ion channels in the plasma
membrane, as well as by mobilization of Ca2+ from intracellular
stores.
One of the best-studied examples of Ca2+-dependent signal
transduction is the depolarization of excitable cells, such as
those of neuronal, cardiac, skeletal and smooth muscle tissue,
which is mediated by inward Ca2+ and Na+ currents. The Ca2+
current is attributed to the movement of ions through N-, L-, Pand T-type Ca2+ channels, which are defined both
pharmacologically and by their biophysical properties, including
conductance and voltage sensitivity. Molecular Probes offers
fluorescent analogs of dihydropyridine and verapamil as ligands
for N- and L-type Ca2+ channels. In addition, exist unlabeled and
fluorescent derivatives of ryanodine, a powerful modulator of the
intracellular Ca2+ channels found in the sarcoplasmic reticulum
and other subcellular organelles.
Fluorescent Dihydropyridines for L-Type Ca2+ Channels: The Ltype Ca2+ channel is readily blocked by the binding of
dihydropyridines to the channel's pore-forming α1-subunit. The
high-affinity (–)-enantiomer of dihydropyridine is available
labeled with either the green-fluorescent DM-BODIPY or the
orange-fluorescent ST-BODIPY fluorophore. DM-BODIPY
dihydropyridine has been employed to investigate the molecular
mechanism for dihydropyridine binding to L-type channels.
Ryanodine Probes for Intracellular Ca2+ Channels: Ryanodine is
a plant alkaloid that mobilizes Ca2+ from intracellular stores by
activating a class of Ins 1,4,5-P3–insensitive Ca2+ channels. It
alters the function of the Ca2+ channel in a complex manner:
submicromolar concentrations lock the channel in a long-lived
open state, whereas micromolar or greater concentrations inhibit
Ca2+ release. BODIPY FL-X ryanodine has been used to
visualize ryanodine receptor distribution in live porcine
endothelial cells, in pancreatic β-cells, in vascular myocytes, in
cardiomyocytes, in frog sympathetic neurons and in the rat
parotid gland.
Probes for Na+ Channels and Carriers
Amiloride Analogs: Probes for the Na+ Channel and the Na+/H+
Antiporter: Amiloride is a compound known to inhibit the Na+/H+
antiport of vertebrate cells by acting competitively at the Na+binding site. The antiport extrudes protons from cells using the
inward Na+ gradient as a driving force. More than 1000 different
amiloride analogs have been synthesized and many of these
tested for their specificity and potency in inhibiting the Na+
channel, Na+/H+ antiporter and Na+/Ca2+ exchanger. We offer
BODIPY FL amiloride a green-fluorescent probe in which the
BODIPY fluorophore is attached at the R3 position.
H2
amiloride
BODIPY® FL amiloride