ARTICLE IN PRESS

Medical Laser Application 24 (2009) 201215

www.elsevier.de/mla

A review of laboratory-based methods to investigate second messengers in

low-level laser therapy (LLLT)
Denise Hawkins Evans, Heidi Abrahamse
Laser Research Group, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein,
Johannesburg 2028, South Africa
Received 24 April 2009; accepted 8 May 2009

Abstract
Second messengers are chemical signals or intracellular molecules (cyclic AMP or inositide) or ions (calcium ions)
that are regulated by extracellular signaling agents such as neurotransmitters and hormones (rst messengers). Second
messengers typically operate by activating protein kinases that phosphorylate various target proteins, thereby altering
the function of these proteins. In order to understand the cellular and molecular responses of cells, for example, in
response to treatment such as low-level laser therapy (LLLT), it is important to determine rstly, if there is an effect on
the intracellular molecules and ions and secondly, if that effect can be directly linked to a change in cellular function
such as cell viability or proliferation. More research is needed to determine the exact mechanism whereby photonic
energy can stimulate injured or wounded cells to restore homeostasis and stimulate cell functions such as DNA, RNA
and protein syntheses. This review discusses selected laboratory methods that can be employed to investigate the role
of second messengers in LLLT.
r 2009 Elsevier GmbH. All rights reserved.
Keywords: Biological response; Biostimulation; Cell signaling; Laser; Second messenger

Introduction
Classes of secondary messengers
Second messengers are molecules that relay signals
received at receptors on the cell surface such as the
arrival of protein hormones or growth factors to target
molecules in the cytosol and/or nucleus. Second
messengers are short-lived intracellular signaling molecules where elevated concentrations lead to rapid
alterations in the activity of one or more cellular
Corresponding author. Tel.: +27 11 559 6550;
fax: +27 11 559 6558.
E-mail address: habrahamse@uj.ac.za (H. Abrahamse).

1615-1615/$ - see front matter r 2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.mla.2009.05.003

enzymes. Removal or degradation of the second

messenger terminates the cellular response. There are
major classes of second messengers namely: (i) hydrophilic, cytosolic molecules: cyclic nucleotides such as
adenosine 3,5 -cyclic monophosphate (cAMP), guanosine 3,5 -cyclic monophosphate (cGMP) and calcium
ions (Ca2+), (ii) hydrophobic molecules: membraneassociated molecules such as inositol triphosphate (IP3)
and 1,2-diacylglycerol (DAG), and (iii) gases such as
nitric oxide (NO) or carbon monoxide (CO). adenosine
3,5 -cyclic monophosphate (cAMP), guanosine 3,5 cyclic monophosphate (cGMP) and Ca2+ are watersoluble molecules located within the cytosol while NO
and CO are both gases that diffuse through the cytosol
and across cellular membranes (Table 1). DAG is an

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Table 1.

D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

Summary of secondary messengers and their interrelated functions.

Messenger

Function

Method

Principle

cAMP
Generated from ATP,
diffuses freely in
cytoplasm

Phosphorylation of cellular proteins.

Regulation of gene transcription
factors.

cAMP EIA

Intensity at 405 nm is inversely

proportional to the [cAMP]

CGMP
Induced respectively by
NO

Regulates activity of cGMP activated

protein kinases.
Phosphorylation of cellular proteins.
Regulates ion channels and inhibits
intracellular calcium uctuations.

cGMP EIA

Intensity at 405 nm is inversely

proportional to the [cGMP]

IP3 and DAG

PIP2 hydrolysis
liberates two second
messengers:
diacyglycerol (DAG)
and inositol
triphosphate (IP3)

DAG remains in the membrane,

where it stimulates protein kinase C
(PKC). IP3 diffuses into the
cytoplasm, where it releases Ca2+
from the endoplasmic reticulum,
which activates Ca2+-dependant
processes.

IP3 Fluorescent
polarization assay
IP3 chemiluminescent
bead assay

The assay is based on competitive

binding between an IP3
uorescent tracer and unlabelled
IP3 from cell lysates or standards

Ca2+
Versatile second
messenger

Regulates many processes; level

controlled by release and removal
(not metabolism).
Direct effect on cell motility.

Fluo 3-AM
Fluo 4-AM
Fura-2AM
QuantiChrom calcium
colorimetric assay

Fluorescent probe for confocal

microscopy, ow cytometry and
microplate screening that
hydrolyzes in the presence of
Ca2+

Nitric oxide (NO)

Activates cGMP
production

Regulated by Ca2+ or calmodulin

and phosphorylation.
Novel mediator of gene regulation
during wound healing.

Nitric oxide activity kit

Nitrate (NO
3 ) converted to
nitrite (NO
2 )+Griess
reagent purple azo compound
(A540 nm)

Carbon monoxide (CO)

Diffuses through
cytosol and across
cellular membranes

CO has been shown to increase

cGMP levels by activating guanylate
cyclase.

Spectrophotometric
detection of CO in cell
culture supernatants

The amount of pure CO was

calculated+expressed as
micrograms per liter total
supernatant

intracellular messenger that remains attached to the

inner membrane and accumulates transiently in cells
exposed to growth factors or other stimuli while IP3
diffuses to the endoplasmic reticulum, where it triggers
release of Ca2+ ions into the cytosol. The released Ca2+
and DAG activate protein kinase C (PKC).
Environmental and hormonal signals regulate various
physiological processes via signal transduction pathways. Essential components of signal transduction
pathways include second messengers. Vital roles for
the second messengers Ca2+, cAMP, cGMP, IP3 and
DAG were rst discovered in animal systems [1,2]. It is
known that a number of chemical and physical stimuli
mediate their effects via transient increases in the
concentration of intracellular free Ca2+ [3]. Receptor
activation, at least in mammalian cells, can trigger the
phosphoinositide cascade, which leads to the production
of IP3 and DAG. IP3 can then provoke the release from
the internal Ca2+ stores by opening intracellular Ca2+

channels [4,5]. Increase in [Ca2+]cyt produced either by

inux or release from internal Ca2+ stores stimulates the
phosphorylation of proteins within the cell. There are
phosphatases that degrade cGMP and IP3 therefore the
concentration of these compounds remains high as long
as they are being synthesized, which in turn depends on
the continued presence of the extracellular signal.
Nitric oxide is produced, from arginine by the enzyme
NO synthase, and once produced, can diffuse through
the membrane of the cell that makes it and into nearby
cells because it crosses membranes readily. NO is very
labile and thus is quite rapidly inactivated by reactions
with water or O2, so it has a lifetime of, at most, a few
seconds. It has recently been shown that the way NO
works on its target cells is to activate a soluble, not
membrane-associated enzyme, guanylate cyclase (GC)
to make cGMP (the second messenger for NO).
An activated receptor binds to a G protein which
activates an enzyme, namely phospholipase C which

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D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

cuts the phospholipid phosphatidyl inositol 4-5 biphosphate (PIP2) to release inositol 1,4,5-triphosphate
(InsP3 or IP3) into the cytosol. IP3 is highly charged
and soluble but DAG remains in the lipid bilayer. The
InsP3 receptor is a Ca2+ selective transmembrane
protein or calcium channel. IP3 binding to its receptor
causes a rapid increase in [Ca2+] in the cytosol so Ca2+
can act as a second messenger molecule for many
different cellular processes [6].
cGMP seems to regulate a second form of Ca2+
channel in the endoplasmic reticulum, called the ryanodine receptor. cGMP activates an enzyme called ADP
ribosyl cyclase, which acts on NAD+ to form cyclic ADP
ribose, which allows calcium to ow into the cytoplasm
and raise the concentration intracellularly. cGMP can be
used in microinjection experiments and in vitro enzymological studies of phosphorylation. The cGMP assay may
also be useful in the determination of cGMP-dependent
protein kinase substrates and inhibitors.
cAMP is generated from adenosine triphosphate
(ATP) and is responsible for the phosphorylation of
cellular proteins and functions to regulate gene transcription factors. Caffeine, theophylline, the cholera
toxin and pertussis toxin all affect second messenger
pathways in cells by affecting the cAMP system. One
way to determine what the second messenger is for a
system is to nd out whether a substance or environmental signal can interfere with or stimulate a response.

Low-level laser therapy (LLLT) and secondary

messengers
LLLT refers to the modality of applying low energy
or low level laser to tissue that stimulates cellular
processes and thereby enhances biochemical reactions.
Some of the medical applications of LLLT include:
acceleration of wound healing, remodeling and repair of
bone, restoration of normal neural function following
injury, pain attenuation and modulation of the immune
system. LLLT or the use of lasers of low power is a
technique currently used to promote wound healing and
provide pain relief. The heliumneon (HeNe) laser at a
wavelength of 632 nm which transmits visible red light
was one of the rst lasers utilized in LLLT. The majority
of work done has been done with infrared semiconductor diode lasers such as the galliumarsenide laser
(GaAs) and the galliumaluminumarsenide (GaAlAs)
laser at wavelengths of 904 and 820 nm or 830 nm,
respectively. All LLLT techniques use radiation with
powers of less than 1 W. LLLT irradiation includes
wavelengths of between 500 and 1100 nm and typically
delivers 10200 mW of power during treatment. LLLT
treatments typically deliver power densities ranging
from 0.05 to 5 W/cm2 and energy densities ranging from
0.5 to 10 J/cm2. This technique utilizes a process for

203

wound healing known as photonic biostimulation.

Many theories exist as to the mechanism of action for
LLLT but simply put, photonic energy is absorbed by
photon acceptor sites on the cell membrane which
increases ATP production and membrane perturbation
to lead to permeability changes, which will trigger a
secondary messenger to initiate a cascade of intracellular
signals that initiate, inhibit or accelerate biological
processes such as wound healing, inammation or pain
management. Second messenger activity results in
functional changes such as increased protein synthesis,
increased secretion and motility changes.
According to Oschman [7], the current understanding
of the cellular signaling cascade and amplication is that
the receptors on the cell surface are the primary sites of
the action of low-frequency electromagnetic elds. It is
at this receptor that cellular responses are triggered by
hormones, growth factors, neurotransmitters, pheromones, antigens or even a single photon. Membrane
signals closely associated with the receptors, such as
adenylate cyclases and G proteins, are considered
secondary messengers that couple a single molecular
event at the cell surface to the inux of a huge number of
calcium ions. Calcium ions entering the cell activate a
variety of enzyme molecules and can produce a cascade
of intracellular signals. These enzymes in turn, are
catalysts and since catalysts are not consumed by
reactions they can act again and again amplifying the
responses until calcium levels drop back to pre-stimulation levels [7].
Reactive oxygen species (ROS), the cumulation of
which is induced by direct irradiation of phagocytes, can
activate or deactivate other cells that are not directly
irradiated. In this way, indirect activation (or suppression) of metabolic pathways in non-irradiated cells
occurs. Cooperative action among various cells via
secondary messengers (ROS, cytokines and NO) requires more attention when the mechanisms of LLLT
are considered at the organism level [8]. Perhaps NO
plays a role in activating secondary messengers responsible for the systemic (whole body) effect following laser
irradiation. A possible mechanism connected with the
NOcytochrome c-oxidase complex has been considered.
Direct activation of cells can lead to the indirect
activation of other cells. This occurs via secondary
messengers released by directly activated cells. For
example, ROS produced by phagocytes, lymphokines
and cytokines produced by various subpopulations of
lymphocytes and/or NO produced by macrophages or a
result of NOhemoglobin photolysis of red blood cells
[8]. Lubart et al. [9] distinguished two different responses
of cells following illumination. Firstly, the endogenous
photosensitizers (porphyrins, avins, cytochromes and
NAD) induce ROS with an increase in [Ca2+] and
stimulation of the mitochondrial respiratory chain

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D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

bringing about an increase in ATP production, which

ultimately results in photobiomodulation to restore
homeostasis of stressed or injured cells. The second
response induces antioxidants with the activation of cell
signaling or secondary messengers with a preconditioning process that also results in photobiostimulation.
The most obvious photochemical and photobiological
effects seen in LLLT or biostimulation are due to excitation
of photon absorbing molecules [10]. The stimulation of
cellular ATP production has been suggested as one of the
most important effects of LLLT. Two redox-type reactions
have been used to explain the primary reaction due to
excitation; rstly photo-excitation of certain chromophores
in the cytochrome c-oxidase molecule causes changes in
redox properties and acceleration of electron transfer and
secondly, NO is released from cytochrome c-oxidase and
increases the O2 binding and respiration rate [11]. Karu [12]
proposed that cytochrome c-oxidase and avoproteins like
NADH in the respiratory chain in the mitochondria can act
as photoreceptors (primary reactions). This can cause a
short time activation of the respiratory chain and oxidation
of NADH pool leading to changes in the redox state of
both mitochondria and cytoplasm with changes in the
proton motive force (pmf), mitochondrial transmembrane
potential (DCm), DpH and cellular redox potential (Eh)
which results in the extra synthesis of ATP. There is a rise in
Na+/H+ antiporter in the cytoplasmic membrane, which
causes a short-term increase in pHi and results in a change
in the redox state of the cytoplasm and of the whole cell. An
increase in Na+/H+ antiporter and increase in Na+/K+ATPase results in an increase in cAMP and intracellular
calcium concentration ([Ca2+]i), which stimulates DNA
and RNA synthesis. A cascade of reactions connected with

alteration in cellular homeostasis parameters ([Ca2+]i, pHi,

cAMP, Eh and ATP) is considered as a photosignal
transduction and amplication chain in a cell (secondary
mechanism). More work on the role of second messengers
in low-level laser therapy is warranted. Several irradiation
parameters must be considered for the development of
photorejuvenation (light energy is used to reverse the
process of photo- or sun-induced ageing to the skin)
techniques including wavelength, pulse duration, spot size,
repetition rate and light dose. However, a practical and
reliable method to compare the potential for epidermal
preservation and dermal broblast stimulation of different
laser devices and irradiation parameters is not currently
available [13].

In vitro laboratory methods

Laboratory-based methods can be used to rstly
determine the effect of LLLT on secondary messengers
such as cAMP, Ca2+ and NO, secondly to determine the
association with other cellular responses that are directly
related to the second messenger such as ATP production, mitochondrial membrane potential and cGMP and
thirdly to relate the activity of second messengers to
cellular functions such as changes in viability, proliferation and cell cycle (Fig. 1 and Tables 2a and 2b).

Secondary messenger activity

ATP and cAMP
Due to its fundamental role in cellular energetics,
metabolic regulation and cellular signaling, determination

Fig. 1. Laboratory-based methods can be used to identify the relationship between a second messenger and other directly associated
responses such as: (1) ATP production and cAMP, (2) NO production and cGMP, and (3) intracellular calcium and mitochondrial
membrane potential (MMP). These responses can be used to relate the activity of second messengers to cellular functions such as
changes in viability (ATP and NADH activity or Caspase 3/7 activity for apoptosis), proliferation and changes in the cell cycle.

Table 2a.

Second messengers (laboratory-based methods with a summary of the cell number, total assay time and detection method used).
Method

Assay time Detection

(h)

ATP viability

Cell suspension (1  105/100 mL)

CellTiter Glos

o1

Luminescence

cAMP

Cell lysate (1  107 cells/mL)

Cell lysate (5  104 cells/mL)
Cell lysate (1  105/100 mL)

Horseradish peroxidase (HRP)-labeled cAMP o4

cAMP-Glos
o2
Enzyme immunoassay
o4

A540 nm
Luminescence
A405 nm

Nitric oxide

Cell lysate (1  105/100 mL)

Nitrate reductase and Griess reagent

o1

A540 nm

cGMP

Cell lysate (1  10 /100 mL)

Enzyme immunoassay

o4

A405 nm

Intracellular Ca2+

5  105 or o1  106 cells/mL

Cell lysate (1  106 cells/mL)
2  107 cells/mL
5  105 or o1  106 cells/mL

Fluo 3-AM uorescent intensity (96-well plate)

Calcium No Wash uorescent plate reader
Fluo-3AM and Fluo-4AM
Fluo-4AM (acetoxymethyl ester)
Rhod-2-acetyl ester (rhod-2-AM)
Fura red (calcium indicator decreased
uorescence on binding)

o1
o2
o4
o1
o1
o1

Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission

Mitochondrial membrane
potential (MMP)

5  105 or o1  106 cells/mL

Fluorescent microscopy:
 Healthy cells Rhodamine
 Healthy cells Texas red
 Unhealthy or apoptotic FITC
For a plate reader (uorescent intensity):
 Healthy cells
 Unhealthy cells (apoptotic)

o1
o1
o1

Excitation/emission at 540/570 nm (orange)

Excitation/emission at 590/610 nm (red)
Excitation/emission at 485/535 nm (orange)

o1
o1

Excitation and emission at 560 and 595 nm

Excitation and emission at 485 and 535 nm

5  105 or o1  106 cells/mL

at
at
at
at
at
at

488/522 nm
450490/540 nm AF40
505/530 nm
490/515 nm (red)
555/590 nm (orange)
436500/640 nm (red)

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Cell number or culture medium

D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

Response

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Table 2b.

Functional changes (laboratory-based methods with a summary of the cell number, total assay time and detection method used).
Method

Assay
Detection
time (h)

Cell morphology

5  105 or o1  106 cells/mL

Light microscopy
Fluorescent probe for microscopy or ow cytometry:
 7-amino-Actinomycin D (DNA)
 Acridine orange (cell cycle): DNA
 Acridine orange (cell cycle): RNA
 Hoechst 33342 (DNA)
 DAPI (DNA and RNA)
 Propidium iodide (DNA vs. RNA)
 Ethidium bromide (nucleic acids)
 DiOC6 (mitochondrial marker)
Fluorescent probe for microscopy or ow cytometry:
 1,10 -dioctadecyl-3,3,30 30 -tetramethyl indocarbocyanine
perchlorate, DiI (lipophilic membrane marker)
 N-(3-triethylammoniumpropyl)-4(dibutylaminostyryl)pyridinium dibromide, FMI-43
(plasma membrane only)
 Tetramethylrhodamine, TMRE (membrane potential)
 Rho123 (membrane potential)
 JC-1 (membrane potential)
 NBD C6-ceramide (golgi marker)
 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolyl
carbocyanin iodide (mitochondrial activity)

o1

Trypan blue exclusion test

o1
o1
o1
o1
o1
o1
o1
o1

Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission

o1

Excitation/emission at 547/565 nm (orange)

o1

Excitation/emission at 488/565 nm (red)

o1
o1
o1
o1
o1

Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission
Excitation/emission

5  105 or o1  106 cells/mL

Viability

o20,000 cells/well in a 96-well plate

5  105 or o1  106 cells/mL
5  105 cells/mL or 50 mg/lane
Cell lysate (1  105/100 mL)
5  105 cells/mL or 50 mg/lane
5  105 or o1  106 cells/mL

Proliferation

Adherent 1  105 cells/well

1  106 cells/mL
1  106 cells/mL
1  106 cells/mL
Cultured cells on sterile cover slip
Adherent 1  105 cells/well
5  104 cells/well (96-well plate)
2  105 cells/mL
5  105 cells/mL or 50 mg/lane

Light microscopy:
Apoptosis:
 Caspase 3/7 activity
 Caspase 3 uorogenic substrate
 Cytochrome c: anti-cytochrome FITC conjugated

 Cytochrome c TiterZyme EIA

 Bcl-2 and Bax (apoptosis)
Propidium iodide (impermeant nucleic acid stain)

at
at
at
at
at
at
at
at

at
at
at
at
at
at

570/610 nm
502/525 nm (green)
460 (blue)/650 nm (red)
350/461 nm (blue/cyan)
358/461 nm (blue/cyan)
488/590 nm (red)
510/595 nm (red)
484/501 nm (green)

549/573 nm (red)
505/533 nm (green)
514/529 or 590 nm (green)
466/536 nm (green)
585/590 nm (red)
510/527 nm (green)

Trypan blue exclusion test

o3
o1
o3
o6
o6
o1

Luminescence
Excitation/emission at 380/438 nm
Western blotting or green-uorescent Alexa Fluor
488labelled secondary antibody
Enzyme immunometric assay
Western blotting or immunohistochemistry
Excitation/emission at 536/617 nm (red)

o24
o24
o4
o6
o24
14
o5
o3

A570 nm (correction at A650 nm)

A570 nm
A450 nm
Light microscopy
A450 nm
Excitation/emission at 530570/580620 nm (pink)
A450 nm
Western blotting or immunohistochemistry

Mitochondrial activity:

 MTT
 XTT
 WST-1
BrdU immunohistochemistry
BrdU cellular ELISA
NADH: resazurin to resorun
NAD+/NADH quantication
Cell cycle: Cdc6 monoclonal antibody

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Cell number or culture medium

D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

Response

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D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

of intracellular ATP (adenosine 50 -triphosphate) is an

irreplaceable tool in the characterization of cellular
physiology [14]. ATP has several metabolic functions as
being involved in phosphorylation reactions, allosteric
regulation, assembly of several cofactors including RNA
and metabolic control by covalent modication [15,16].
ATP can be measured by bioluminescence using an
assay that is based on luciferases requirement for ATP
in producing light [15,17,18]. The CellTiter-Glos
luminescent cell viability assay (Promega, G7570) is
based on the quantitation of ATP present, which signals
the presence of metabolically active cells or viable cells.
The assay uses a unique, stable luciferase reaction where
the amount of light produced is proportional to the
amount of ATP present, which reects the number of
viable cells or metabolically active cells. Briey, an equal
volume of reconstituted CellTiter-Glos reagent is added
to 50 mL of cell suspension (1  105 cells/50 mL) and
mixed for 2 min to induce cell lysis. The suspension is
incubated at room temperature for 10 min to stabilize
the luminescent signal, which is recorded using a
luminometer [17,1921]. ATP standard curves are
prepared with different concentrations of ATP ranging
from 20 to 320 mM. Each sample is assayed in triplicate
and a mean value7SD of ATP content is expressed and
calculations are made against the curve to determine the
cellular ATP level, which is expressed as nmol/cells [22].
Adenosine 30 ,50 -cyclic monophosphate (cAMP) is a
ubiquitous second messenger involved in various cellular activities that is converted from ATP via adenylyl
cyclases (AC). Ca2+ and its downstream effectors
are also regulators of AC activity. Depending on the
AC subtype, this regulation might be stimulatory or
inhibitory [23]. A large family of cyclic nucleotide
phosphodiesterases regulates the levels of cAMP and
cGMP [24]. cAMP may affect cellular function through
several different mechanisms including the activation of
cAMP-dependent protein kinase (PKA), guanine nucleotide exchange factors (GEFs), and cyclic nucleotidegated (CNG) channels. It may also be extruded by
certain cell types and have extracellular roles as well.
GEFs facilitate the exchange of GDP for GTP and,
therefore, promote the activity of G proteins. Exchange
protein activated by cAMP (Epac) 1 and 2 are GEFs
activated upon binding to cAMP. CNG channels are
cation channels activated by cGMP and/or cAMP.
These channels regulate membrane potential, and due
to their Ca2+ permeability, can alter the levels of
intracellular Ca2+.
Briey, the cAMP assay is based on the competitive
binding technique in which cAMP present in a sample
(cell lysate of 1  107 cells/mL) competes with a xed
amount of horseradish peroxidase (HRP)-labeled cAMP
for sites on a mouse monoclonal antibody (Parameter,
KGE002). During 3 h incubation, the monoclonal antibody becomes bound to the goat anti-mouse antibody

207

coated onto the microplate while the cAMP conjugates

and samples complete for binding sites. Following a
wash to remove excess conjugate and unbound sample,
a substrate solution (hydrogen peroxide and stabilized
chromogen or tetramethylbenzidine (TMB)) is added to
the wells to determine the bound enzyme activity. The
color development is stopped (2 N H2SO4) after 30 min
and the absorbance is read at 450 nm (wavelength
correction at 540 nm). The intensity of the color is
inversely proportional to the concentration of cAMP in
the sample.
The cAMP-Glos assay (Promega, V1501) is a homogeneous, bioluminescent and high throughput assay to
measure cAMP levels in cells. Cells (5  104 cells/mL)
are lysed to release cAMP and the cAMP-Glos detection
solution, which contains protein kinase A, is added.
The assay is based on the principle that cAMP stimulates
protein kinase A (PKA) holoenzyme activity, decreasing
available ATP and leading to decreased light production
in a coupled luciferase reaction. The Kinase-Glos
reagent is then added to terminate the PKA reaction
and detect the remaining ATP via a luciferase reaction.
Luminescence can be correlated to the cAMP concentration using a cAMP standard curve [25,26].
Nitric oxide and cGMP
NO is a gaseous free radical with a short half-life in
vivo of a few seconds or less. Therefore, the levels of the
more stable NO metabolites, nitrite (NO
2 ) and nitrate
(NO
3 ), have been used in the indirect measurement of
NO in biological uids [27]. Altered levels of NO have
been shown to be associated with sepsis, reproduction,
infection, hypertension, exercise, type-2 diabetes, hypoxia, and cancer [2831]. Because it is lipid soluble, NO
is not stored but is synthesized de novo and diffuses
freely across lipid membranes. NO has the potential to
mediate its effects on target cells via several different
mechanisms. For instance, NO-mediated activation
of the enzyme guanylate cyclase (GC) catalyzes the
formation of the second messenger guanosine 30 ,50 -cyclic
monophosphate (cGMP). NO also functions as an antitumor and anti-microbial agent via mechanisms that
include its conversion to peroxynitrite (ONOO), the
formation of S-nitrosothiols, and the depletion of
arginine [32]. Another putative role for NO includes
the suppression of mitochondrial respiration through
the inhibition of cytochrome oxidase [33]. NO may
also modify protein activity through post-translational
nitrosylation via the attachment of an NO moiety to the
thiol side chain of cysteine residues [3436].
NO is a major mammalian secretory product that
initiates host defense, homeostatic and developmental
functions by either direct effect or intracellular signaling. The transient and volatile nature of NO makes
it unsuitable for most convenient detection methods;
however, two stable breakdown products, NO
2 and

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D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

NO
3 can be easily detected by photometric means as a
quantitative measure of NO production. The measurement of total nitrate/nitrite involves a simple two-step
process. The rst step is to convert nitrate to nitrite
utilizing nitrate reductase. The second step involves the
addition of the Griess reagent (sulfanilamide in 2 N
hydrochloric acid and N-(1-Naphthyl) ethylenediamine
in 2 N hydrochloric acid), which converts nitrite into a
deep purple azo compound that absorbs visible light at
540 nm (wavelength correction at 690 nm). The measurement of the absorbance of the azo chromophore
accurately determines the total nitric oxide production.
The detection limit of the reaction is 1 mM nitrite,
which translates to approximately, 2.5 mM nitrate in the
original sample. The relative levels of nitrite and nitrate
can vary substantially; therefore the most accurate
determination of total nitric oxide production requires
quantication of both nitrate and nitrite (Assay Designs
correlate NO2/NO3 assay kit, 917-010 or Parameter
total NO/nitrite/nitrate assay, KGE001).
Guanosine 30 ,50 -cyclic monophosphate (cGMP) has
been shown to be present at levels typically 10100-fold
lower than cAMP in most tissues and is formed by the
action of the enzyme GC on GTP [37]. cGMP is
implicated with a range of biological functions such as
regulating smooth muscle contractility, cell survival,
proliferation, axon guidance, synaptic plasticity, inammation, angiogenesis, and the activity of CNG channels
[38,39]. Stimulators of GC such as sodium nitrate and
NO stimulate cGMP levels. NO can be synthesized from
L-arginine and diffuse through cell membranes [40,41].
The interaction of NO with GC allows cGMP to act as a
third messenger in some cells [42]. Briey, the determination of cGMP uses a polyclonal antibody to cGMP to
bind, in a competitive manner, the cGMP in a standard
or 0.1 M HCl-treated sample (cell lysate) that has an
alkaline phosphatase (ALP) molecule covalently attached to it (Assay Designs correlate EIA direct cGMP,
900-014). After a short incubation at room temperature
for 2 h the excess reagents are washed away and alkaline
phosphatase (r-nitrophenyl phosphate or pNpp) substrate is added and incubated at room temperature for
1 h. The reaction is stopped and the yellow color
generated is read on a microplate reader at 405 nm.
The intensity of the bound yellow color is inversely
proportional to the concentration of cGMP. The
measured optical density is used to calculate the
concentration of cGMP. The same method can be used
to determine cAMP instead a polyclonal antibody to
cAMP is used to bind the cAMP in a competitive
manner (Assay Designs correlate EIA direct cAMP,
900-066).
Ca2+ and mitochondrial membrane potential
Mitochondria possess an effective Ca2+ transporting
system driven by the mitochondrial membrane potential

[43]. Mitochondrial membrane potential (MMP) and

intracellular calcium concentration ([Ca2+]i) play an
important role in the mechanism of apoptosis induced
by oxidative stress. However, it is unknown what
the relationship between MMP and [Ca2+]i has
upon oxidative stress, and their individual modication
by medication or therapy. The intracellular Ca2+
stores play a very important role in Ca2+ signaling
and uctuations in membrane potential lead to
changes in basal cytosolic free Ca2+ concentration
[Ca2+]cyto [44,45].
Changes in intracellular Ca2+ concentration can be
monitored using a single wavelength uorescent probe,
Fluo-3AM. Briey, cells are loaded for 30 min at 25 1C
with 5 mM Fluo-3AM containing 1 mM pluronic acid
F-127 for proper dispersal and 0.25 mM sulnpyrazone,
an organic anion transport inhibitor to reduce leakage
of the Fluo-3AM dye. Just before use, cells are washed
with medium to remove non-hydrolyzed Fluo-3AM.
Fluorescence measurements are performed at 25 1C at
an excitation of 488 nm and emission of 522 nm. To
convert uorescence values into absolute [Ca2+]i,
calibration is performed at the end of each experiment.
[Ca2+]i is calculated using the equation [Ca2+]i Kd
(FFmin)/(FmaxF), where Kd is the dissociation constant of the Ca2+-Fluo-3AM complex (400 nM), and
F represents the uorescence intensity of the cells. Fmax
represents the maximum uorescence (obtained by
treating the cells with 10 mM calcium ionophore), and
Fmin corresponds to the minimum uorescence (obtained
from ionophore-treated cells in the presence of 3 mM
EGTA). Fluorescence intensities are expressed as the
increase in uorescence with respect to baseline uorescence intensity before stimulation [17].
The Calcium No Wash (DiscoveRx, 90-0080 L) is a
homogenous assay that provides an alternative method
to traditional wash and quench-based assays for the
detection of format calcium mobilization and changes in
intracellular calcium in response to G protein-coupled
receptor (GPCR) activation. GPCRs are transmembrane spanning proteins that transduce an extracellular
signal (ligand binding) into an intracellular event
(G-protein activation). Cells expressing a GPCR of
interest that signals through calcium are pre-loaded
with a calcium-sensitive dye and then treated with a
compound. Upon stimulation, the receptor signals the
release of intracellular calcium, which then causes the
dye to uoresce. The signal generated in the assay
is measured using a uorescent plate reader that is
capable of detecting changes in uorescent intensity with
excitation of 450490 nm and emission of 540 nm AF40.
The assay has a linear detection range of 2080 mg/dL
[46]. BioAssay Systems calcium assay kit can also be
used to measure calcium directly in biological samples
without any pre-treatment. A phenolsulphonephthalein
dye forms a stable blue-colored complex specically

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with free calcium. The intensity of the color, measured

at 612 nm, is directly proportional to the calcium
concentration in the sample. The assay can detect
between 0.08 mg/mL (20 mM) and 20 mg/mL (5 mM)
Ca2+ in a 96-well plate assay (BioAssay System
QuantiChrom calcium assay, DICA-500).
Fluo-3AM is a cell-permeable uorescent indicator of
intracellular calcium levels. It crosses the cell membrane
due to its AM ester structure. Esterases in the cell then
hydrolyze the AM ester to yield Fluo 3. This compound
is non-uorescent until associated with Ca2+. The lower
binding afnity of this compound allows measurement
of higher peaks of Ca2+ and has been used to detect
photochemically generated cytosolic calcium pulses.
Detailed procedures for the use in measuring cytosolic
Ca2+ in platelets and neutrophils in the presence of
plasma have been reported. Briey, 2  107 cells/mL are
loaded by incubation at 37 1C for 30 min with 2 mM
Fluo-3AM. The cells are washed twice and resuspended
to 2  106 cells/mL in Ca2+-free medium (145 mM
NaCl, 1 mM Na2HPO4  2H2O, 0.5 mM MgSO4  7H2O,
5 mM glucose, 20 mM Hepes, pH 7.4) to which
1 mM CaCl2 is added as required. Fluorescence is
measured at 505 nm excitation and 530 nm emission.
Fluo-3AM (Sigma, F6142) is non-uorescent until
it is hydrolyzed intracellularly and/or in the presence
of Ca2+. Fluo-4AM (Sigma, 93596) is an improved analogue of the popular calcium indicator
Fluo-3AM and is the preferred indicator for confocal
microscopy, ow cytometry and microplate screening
applications.
To detect changes in mitochondrial and cytosolic
calcium induced by stimulation with the purinergic
receptor ligand ATP cells were seeded in 4-well
chambered coverglass slides and imaged using a Zeiss
Axiovert 200 M microscope. Samples were excited with
bandpass lters at an excitation of 490 nm and emission
of 515 nm for Fluo-4AM (Invitrogen, F14217) and an
excitation of 555 nm and emission of 590 nm for rhod-2acetyl ester (rhod-2-AM), an indicator of mitochondrial
calcium. Images were collected every 15 s for 30 min.
A minimum of 500800 cells were imaged per treatment
group for studies of ATP-induced changes in Fluo-4 and
rhod-2 uorescence across 23 independent experiments
[47]. Furthermore, changes in intracellular Ca2+ could
be measured by uorescent microscopy using Fura2AM with a uorescence ratio at 340380 nm. The
changes in intracellular Ca2+ could be visualized as
color images and the [Ca2+]i could be determined using
10 mM Fura-2-containing buffer solution with 1 mM
EGTA and various concentrations of Ca2+ from 20 to
500 nM of CaCl2 [4850].
Mitochondrial transmembrane potential (DCm) is an
important parameter of mitochondrial function and has
been used as an indicator of cell health. Several key
elements occur in mitochondria, including the release of

209

caspase activators such as cytochrome c, changes in

electron transport and loss of MMP. Changes in MMP
reected by different forms of JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-benzimidazol-carbocyanine
iodide) as either green or red uorescence can be both
qualied and quantied by uorescent microscopy, ow
cytometry, a uorescence plate reader with appropriate
lter set or confocal laser scanning microscopy [51]. JC1 is a cationic mitochondrial vital dye that is lipophilic
and becomes concentrated in the mitochondria. The
accumulation is proportional to the membrane potential; the more dye that accumulates in mitochondria the
greater the DCm and ATP-generating capacity [17]. JC-1
has advantages over other cationic dyes in that it can
selectively enter the mitochondria and reversibly change
from green to red as the membrane potential increases.
In healthy cells with high mitochondrial DCm, JC-1
spontaneously forms complexes known as J-aggregates
with intense red uorescence. On the other hand, in
apoptotic or unhealthy cells with low DCm, JC-1
remains in a monomeric form, which shows only green
uorescence. For uorescent microscopy, healthy cells
with mainly JC-1 aggregates can be detected with
uorescence settings usually designed to detect rhodamine (excitation/emission at 540/570 nm) or Texas red
(excitation/emission at 590/610 nm). Apoptotic or unhealthy cells with mainly JC-1 monomers can be
detected with settings designed to detect uorescein
isothiocyanate (FITC) (excitation/emission at 485/
535 nm). For a plate reader, healthy cells with mainly
JC-1 aggregates show strong uorescent intensity with
excitation and emission at 560 and 595 nm, respectively.
In apoptotic or unhealthy cells, JC-1 monomers show
strong uorescence intensity with excitation and emission at 485 and 535 nm, respectively (Cayman JC-1,
1009172).
The JC-1 dye allows the identication of populations
with different mitochondria morphology as well as the
functionality of this organelle in cells incubated for 1, 6
and 24 h after irradiation with a low power laser. The
results may reect a biostimulative boost that causes a
shift of the cell from a quiescent to an activated stage in
the cell cycle heralding proliferation and suppression of
inammation. Melo et al. [52] used serum to analyze
mitochondrial function after laser treatment using a
mitochondrial respiratory function assay and changes in
membrane potential.
Rhodamine 123 (Rh123) is a cell-permanent, cationic,
uorescent dye that is readily sequestered by active
mitochondria without inducing cytotoxic effects. The
stained mitochondria appear yellow-green when viewed
through a uorescein long-pass optical lter or can be
sorted using ow cytometry. Rhodamine 123 (10 mg/mL)
can be used to study apoptosis, mitochondrial enzyme
activity and mitochondrial transmembrane potential.
Damage to the mitochondria results in reduced uptake

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D.H. Evans, H. Abrahamse / Medical Laser Application 24 (2009) 201215

of Rh123 and thus serves as an assay for mitochondria

transmembrane potential. Rh123 can also be used to
detect the early stages of oxidative stress and apoptosis
in cells, which are associated with increased Rh123
uorescence [53].

Functional changes resulting from the activation of

secondary messengers
Cell morphology
Cellular energy produced during mitochondrial respiration is stored as an electrochemical gradient across
the mitochondrial membrane. This accumulation of
energy in healthy cells creates a mitochondrial transmembrane potential, called delta-psi or DCm that
enables the cell to drive the synthesis of ATP. Disruption of DCm has been shown to be one of the rst
intracellular changes following the onset of apoptosis.
Fluorescent probes with an ability to selectively
localize in or with a specic afnity for a specic
cellular component can be used for microscopy.
Examples include actinomycin D, acridine orange,
Hoechst 33342 [54], DAPI [51], propidium iodide and
ethidium bromide for nucleic acids; DiO, DiI [55], FMI43 for proteins, plasma membranes and endoplasmic
reticulum; TMRE and JC-1 for membrane potential and
nally NBD C6-ceramide for the Golgi apparatus
(Table 2b).
Cells can be stained for microscopic observation or
for ow cytometry. A DePsipher assay (Assay Designs,
900-167) uses a lipophilic cation (5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolyl carbocyanine iodide)
which can be used as a mitochondrial activity marker.
The dye aggregates upon membrane polarization forming an orange-red uorescent compound. If the potential
is disturbed, the dye cannot access the transmembrane
space and remains or reverts to its green monomeric
form. The red aggregates have absorption/emission
maxima of 585/590 nm and the green monomers of
510/527 nm. In ow cytometry experiments, the green
monomer can be detected using a uorescein channel
(FL1) and the red aggregates can be detected using the
propidium iodide channel (FL2).

Cell viability, apoptosis, and necrosis

Cell viability is detected by trypan blue exclusion
assay. Cells are stained with 0.4% trypan blue in
phosphate buffered saline (PBS) followed by examination with a hemocytometer under an inverted microscope [21,56]. Cells that exclude the dye are considered
viable and the results are expressed as percentage trypan
blue exclusion (% TBE). In parallel, cells are stained
with propidium iodide (1 mg/mL) in binding buffer. The
cells are washed in cold PBS and counted with a

hemocytometer. Cells that stain with red, propidium

iodide are considered necrotic [57].
Intracellular lactate dehydrogenase (LDH) can also
be used to detect changes in cell membrane integrity,
cell-mediated cytotoxicity, cytotoxicity mediated by
other agents and total cell number. LDH leakage is
well known as an indicator of cell membrane integrity
and cell viability. LDH is a stable cytosolic enzyme that
is released into the culture medium upon cell lysis. LDH
in culture supernatants converts a tetrazolium salt (INT)
into a formazan product. The color formed is proportional to the number of lysed cells (Promega, G1780).
A decrease in MMP is an early event during apoptosis
and cytochrome c released from the mitochondria
triggers apoptosis. The protein is located in the space
between the inner and outer mitochondrial membranes
and can be detected by ELISA [57]. An apoptotic
stimulus triggers the release of cytochrome c from the
mitochondria into the cytosol where it binds Apaf-1.
The cytochrome c/Apaf-1 complex activates caspase-9
(Promega, 8210), which then activates caspase-3 (Promega, G8090). Cytochrome c released from mitochondria into cytosol can be determined by Western blotting
using a cytochrome c-antibody (BioVision, K257-100)
or by enzyme immunometric assay (EIA) using cell
lysates (Assay Designs human cytochrome c EIA, 900141). The Caspase-Glos 9 and Caspase-Glos 3/7 assays
are homogeneous luminescent assays that measure
caspase-9 activity and caspase-3/7 activity, respectively
[58]. The assay provides a proluminescent caspase
DEVD-aminoluciferin substrate in a buffer system
optimized for caspase activity, luciferase activity and
cell lysis. Addition of the single Caspase-Glos reagent
in an add-mix-measure format results in cell lysis,
followed by caspase cleavage of the substrate. This
liberates free aminoluciferin, which is consumed by the
luciferase, generating a glow-type luminescent signal.
The luminescent signal generated is proportional to the
amount of caspase activity present [21,59,60]. Caspase-3
activity can be measured using a caspase uorogenic
substrate (Ac-DEVD-AMC) that liberates uorescence
(AMC), which is measured using a spectrouorometer
(excitation 380 nm, emission 438 nm) [61].
Cell proliferation
A number of methods have been developed to study
cell viability and proliferation in cell populations. The
most convenient modern assays have been developed in
a microplate format (96-well plates). Colorimetric assays
allow many samples to be analyzed rapidly and
simultaneously and samples can be measured directly
in the microplate with an ELISA plate reader.
Cellular damage will inevitably result in loss of the
ability of the cell to maintain and provide energy for
metabolic cell function and growth. Metabolic activity
assays usually measure mitochondrial activity and the

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cells are incubated with a colorimetric substrate (MTT,

XTT, WST-1). The major advantage of these compounds is that only metabolically active or viable cells
are able to convert them (XTT and MTT) to a watersoluble formazan salt. After it is solubilized, the
formazan formed can easily and rapidly be quantied
in a conventional ELISA plate reader at 570 nm. The
microtiter plate is now widely used to quantify cell
viability, cell proliferation and cytotoxicity. For the
WST-1 assay (Roche, 11 644 807 001), cells are cultured
in a 96-well microplate with WST-1 for approximately
0.55 h. During the incubation period, viable cells
convert WST-1 to a water-soluble formazan dye. The
formazan dye is quantied with an ELISA plate reader
and the absorbance at 450 nm directly correlates with
the cell number.
During the S-phase the cell undergoes DNA synthesis
and replicates its genome. If labeled DNA precursors
(5-bromo-20 -deoxyuridine (BrdU)) are added to the cell
culture, cells that are about to divide incorporate BrdU
into their DNA. The incorporated BrdU can be detected
by a quantitative cellular enzyme immunoassay (Roche,
11669915001) or by immunohistochemical staining
using the immunoenzyme HRP method (US Biological,
B2851) with monoclonal antibodies against BrdU.
Experiments have shown that the thymidine analogue
BrdU is incorporated into cellular DNA like thymidine
to give evidence of DNA replication. The use of BrdU
for proliferation assays circumvents the disadvantages
associated with the radioactive compound [3H]-TdR.
Abe et al. [62] used the BrdU incorporation assay to
detect DNA synthesizing cells. Incorporation of BrdU
into newly synthesized DNA permits indirect detection of
rapidly proliferating cells with a biotinylated monoclonal
anti-BrdU antibody thereby facilitating the identication
of cells that have progressed through the S-phase of the
cell cycle during the BrdU labeling period [61,63]. In
immunohistochemistry, streptavidin-peroxidase is used as
a signal generator and diaminobenzidine (DAB) in the
presence of hydrogen peroxide is used as a chromogen,
staining BrdU-incorporated nuclei dark brown.
BrdU-ELISA assay is based on the incorporation
of the pyrimidine analogue BrdU into the DNA of
proliferating cells that are cultured in microtiter plates
[64]. After its incorporation into DNA, BrdU in the cell
is detected by anti-BrdU monoclonal antibody and
horseradish peroxidase-conjugated goat anti-mouse immune globulin (IgG) is added, which binds to the
detector antibody. The horseradish peroxidase catalyzes
the conversion of the chromogenic substrate tetramethylbenzidine (TMB) from a colorless solution to a
blue solution (or yellow after the addition of stopping
reagent). The color is quantied by spectrophotometry
at 450 nm and reects the relative amount of incorporated BrdU in the cells (CycLexs BrdU Cellular ELISA
Kit, CY-1142).

211

Assay of nicotinamide nucleotides is of continual

interest in the studies of energy transforming and redox
state of cells and tissues. NADH/NAD quantication
provides a convenient tool for sensitive detection of the
intracellular nucleotides: NADH, NAD and their ratio
(Biovision, K337-100). The CellTiter-Blues cell viability
assay (Promega, G8080) provides a homogeneous,
uorescent method for monitoring cell viability. The
assay is based on the ability of living cells to convert a
redox dye (resazurin by NADH) into a uorescent end
product (resorun), which is recorded using a uorometer. Nonviable cells rapidly lose metabolic capacity,
do not reduce the indicator dye, and thus do not
generate a uorescent signal. The absorbance maximum
of resazurin is 605 nm and that of resorun is 573 nm.
Resazurin is dark blue in color and has little intrinsic
uorescence until it is reduced to resorun, which is pink
and highly uorescent at an excitation wavelength of
579 nm and emission of 584 nm [6567]. NADH, NAD
and their ratio is of continual interest in studies of
energy transforming and redox state of cells and tissues.
An assay can be used to specically recognize NADH/
NAD in an enzyme cycling reaction with no requirement to purify NADH/NAD from samples (BioVision,
K337-100).
Cell cycle
In any organism, the rate of cell division is a tightly
regulated process that is intimately associated with
growth, differentiation and tissue turnover. Generally,
cells do not undergo division unless they receive signals
that instruct them to enter the active segments of the cell
cycle. The signals that induce cells to divide are diverse
and trigger a large number of signal transduction
cascades.
The transmission of genetic information from one cell
generation to the next requires the accurate duplication
of the DNA during the S-phase of the cell cycle.
Initiation of DNA replication is a highly regulated
process that requires the ordered assembly of many
proteins at the origin of DNA replication to form a
competent, pre-replicative chromosomal state, as well as
cell cycle regulated protein kinase pathways. Several
proteins required for initiation are suspected to be prereplicative components [68]. Most strongly implicated is
the initiation protein Cdc6. Cdc6 is a downstream target
of molecular signaling pathways that control the G1/S
transition. Cdc6 is only expressed in actively replicating
cells and quiescent cells in G0 do not express the protein,
making it an excellent marker for cell proliferation.
Cdc6 immunolocalization may be used as an index of
cell proliferation in tissue sections. Monoclonal antibody reacting specically with Cdc6 is also a useful tool
in the investigation of the molecular mechanisms that
determine how DNA replication is initiated, how it is
restricted to certain cell cycle phases and how replication

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occurs in cells. Cdc6 antibody (Southern Cross Biotechnology sc-9964) can be used for Western blotting using a
dilution range from 1:100 to 1:1000 with a goat antimouse IgG-HRP secondary antibody (sc-2005, 200 mg/
0.5 mL). The cell cycle can also be analyzed by xing
cells in 70% ethanol, washing twice in PBS containing
10% fetal bovine serum and treating the cells with
RNase (1 U/1  106 cells) for 30 min at 37 1C. The cells
are chilled on ice and stained overnight with propidium
iodide (50 mg/1  106 cells) in the cold. Fluorescent
histograms are obtained on a Coulter cell sorter using
an argon laser and the mean peaks are analyzed into G1,
G2/M and S-phase populations [56,61,69].

Conclusion
LLLT (especially red and near-infrared light) promotes proliferation of multiple cells, which is mainly
through the activation of the mitochondrial respiratory
chain and the initiation of cellular signaling [70]. It has
been reported that the DCm/ATP/cAMP/JNK/AP-1
and ROS/Src pathways are involved in laser-induced
proliferation [70]. Recently, a large number of signaling
proteins were identied as having important roles in the
process of LLLT-induced effects [70]. Gao and Xing [70]
specically referred to certain lasers and the link to cell
signaling such as (i) GaAs 904 nm with complexes II and
IV of the mitochondrial respiratory chain, (ii) HeNe
632.8 nm with the tyrosine protein kinase receptor
(TPKR) and the MAPK/ERK pathway, (iii) HeNe
632.5 nm with increased NO secretion and eNOS
expression, (iv) green laser 532 nm with increased
DCm, (v) light at 780 nm with increased DCm and
increased expression of interleukin-1 alpha (IL-1a) and
interleukin-6 (IL-6), (vi) HeNe 632.8 nm with increased
activation of PKCs, (vii) HeNe laser 632.8 nm with
increased DCm, ATP, cAMP and the uptake of Ca2+ by
mitochondria, (viii) HeNe laser 632.8 nm with increased
intracellular ROS, (ix) HeNe laser 632.8 nm with
increased expression of cell-cycle regulatory proteins:
cyclin D1, cyclin E and cyclin A, and (x) GaAlAs
650 nm with reduced expression of TNF-a, a potent proinammatory cytokine [70]. LLLT induces the synthesis
or release of many molecules such as growth factors,
interleukins and inammatory cytokines; however,
studies provide evidence that the signaling proteins,
some of which are regulated by mitochondrial signaling,
involved in LLLT-induced effects warrant special
attention [70].
This review identied a number of protocols and
methods to determine the effect of an extracellular
signal, such as a single photon of energy, on the activity
of secondary messengers (cAMP, intracellular calcium
or NO). The review also identied laboratory methods

to determine if there is an association between the

activation of a secondary messenger and a specic
change in intracellular signals (membrane potential,
ATP or cGMP production). Given that the majority of
studies indicate that NO functions in a protective role, it
is important to identify the mechanism responsible for
these protective effects. Using the protocols outlined
above it is possible to identify putative mechanisms such
as an NO-mediated increase in cGMP, an attenuation of
calcium accumulation, a decrease in oxygen consumption, an opening of the mitochondrial ATP-dependent
potassium channel or an inhibition of mitochondrial
permeability transition. Specic methods can be used to
measure the effect on cell function such as changes in
cell morphology, viability, cell proliferation and changes
in cell cycle. This review provides a clear guideline
of the laboratory-based methods available to measure
the effect of an extracellular signal whether a hormone,
growth factor or single photon on the activity of
secondary messengers that initiate, accelerate or inhibit
biological processes. There are many methods and
protocols available to measure the responses discussed
in this review; however, only a few have been selected to
demonstrate the principle and application of each
method. It is important to remember that the selection
of an appropriate method depends on a number of
variables such as the equipment that is available (plate
reader, uorescent microscope and spectrophotometer),
the cost and time implications, the availability and most
importantly the type of response that you want to
measure or the specic application.

Acknowledgement
This study is funded by the University of Johannesburg,
CSIR National Laser Centre (CSIR/NLC) Rental Pool
Program, National Research Foundation of South Africa
and Medical Research Council (MRC) of South Africa.

Zusammenfassung
Labor-basierte Methoden zur Untersuchung von fur die
Low-Level-Lasertherapie (LLLT) relevanter sekundarer
bersicht
Botenstoffe Eine U
Sekundare Botenstoffe sind chemische Signale oder
intrazellulare Molekule (z.B. zyklisches Adenosinmonophosphat (cAMP), Inosite) oder Ionen (z.B. CalciumIonen), die durch extrazellulare Liganden (Signalstoffe)
wie beispielsweise Neurotransmitter und Hormone
(primare Botenstoffe) reguliert werden konnen.
Sekundare Botenstoffe aktivieren typischerweise Proteinkinasen, was zur Phosphorylierung wichtiger Proteine fuhrt und so spezische Zellfunktionen anstot.

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Um die zellulare Wirkung der Laserstrahlung, z.B. bei

der Low-Level-Lasertherapie (LLLT) zu verstehen, ist
es besonders wichtig zunachst zu untersuchen, (1) ob ein
Effekt auf die intrazellularen Molekule und Ionen
besteht und (2) ob dieser Effekt direkt mit einer
nderung der Zellfunktion, z.B. der Lebensfahigkeit
A
oder der Proliferation von Zellen, verbunden ist.
Weitere Forschungsarbeit ist notig, um zu klaren,
durch welche Mechanismen eine niederenergetische
Laserbestrahlung zur Regeneration geschadigter Zellen
beispielsweise durch Starkung der Homoostasis oder der
Steigerung der DNA-/RNA-Synthese beitragt. Der
bersichtsartikel diskutiert ausgewahlte
vorliegende U
Labormethoden, die zur Untersuchung von sekundaren
Botenstoffen, die bei der LLLT eine Rolle spielen,
geeignet sind.
Schlusselworter: Biologische Reaktion; Zellsignalisierung;
Laser; Sekundare Botenstoffe

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