Measurements of Cell Jiability Measurements of Cell Jiability

1ung 1ung- -Keug Park Keug Park

Department of Chemical
& Biochemical Engineering,
Dongguk University
Biomaterials Classification of Biomaterials Classification of
Aew Biotechnology Aew Biotechnology
Cellular components (protein, lipid, enzymes etc.)
Microorganisms
(including recombinant bacterial cells)
nimal cells or tissues
(including human cells and tissue)
Plant cells or tissues
Organs (liver transplantation etc.)
Whole bodies (transgenic animals etc.)
Classification of Classification of
Animal (Human) Cell 1echnologies Animal (Human) Cell 1echnologies
!roduction oI protein therapeutics using large-scale
animal cell culture.
(ex. Mab, E!, BM!, Vaccines etc.)
Use oI animal cells or tissue as a biomass (ex. Bone
marrow cells, bioartiIicial organ technology.
1issue Engineering (1E)
Transplantation oI gene manipulated cells or stem
cells Ior gene therapy or tissue regeneration.
(Upcoming technology)
Cell counting and viability Cell counting and viability
To measure perIormance, to achieve reproducibility, or to
make comparative studies, a means oI quantiIying the cell
population is need.
The growth oI mammalian cells in culture can be
monitored by a number oI parameters related to the
increase oI cellular biomass over time.
The simplest methods is by cell counting at regular
intervals.
The system oI viability assay Ior mammalian cell culture
can be applied to the determination oI cell viability Ior
engineered tissue.
Recent technological advances resulting in improved
maintenance oI cell viability and Iunction in culture and
bioreactor designs have led to the development oI a number oI
bioartiIicial devices.
In an eIIort to determine cell viability, a number oI assays that
correlate with either reproductive or Iunctional capabilities
have been developed.
Viability measurements derived Irom these diIIerent criteria
do not always correlate well with each another. For instance, a
viability test based on membrane integrity judges cells with
Iunctionally undamaged membranes to be viable even iI they
have lost the ability to proliIerate.
Cell Jiability Cell Jiability
Functional assay
DAAlabeling assay
Morphological assay
Reproductive assay
Membrane integrity assay
1he major criteria employed in viability assay 1he major criteria employed in viability assay
Category of
viability assay
ssays Principles
Membrane integrity
assay
-Exclusion dyes
-Fluorescent dyes
-LDH leakage
The determination of membrane integrity via
dye exclusion from live cells
Functional assay
-MTT, XTT assay
-Crystal violet/ cid
phosphatase(P) assay
-lamar Blue oxidation-
reduction assay
- Neutral red assay
-3H]-thymidin/ BrdU
incorporation
Examining metabolic components that are
necessary for cell growth
DN labeling assay -Fluorescent conjugates
Simultaneous cell selection and viability
assay
Morphological
assay
-Microscopic observation
Determination of morphological change
Reproductive assay
-Colony formation assay
Determination of growth rate
Measurement
Characteristics
Methods
Direct
- Distinguish viable
and non-viable cell
- Simple, quick, cheap
- small fraction of the
total cells from a cell
suspension
- Microscopic counting(Hemocytometer)
: Trypan blue, Crystal violet
- Electronic counters
: Coulter counter
Indirect
- Monolayer
- Immobilized in matrix
- Lactate dehydrogenase activity
- Functional assay
- DN labeling assay
Cell counting Cell counting
Membrane integrity assay
Hemocytometer
Trypan blue assay / utomatic trypan blue method
LDH(lactate dehydrogenase) leakage / Fluorescent dyes
Functional Assay
MTT / XTT assay
Crystal violet / cid phosphatase(P) assay
lamar Blue oxidation-reduction assay / Neutral red assay
3H]-thymidin and BrdU incorporation
DAA Assay
Enzymatic DN labeling / DN-binding dye labeling
Morphological assay
Reproductive Assay
Colony-forming efficiency
aser scanning confocal microscopy
Auclear magnetic resonance methods
Membrane integrity assays Membrane integrity assays
Features distinguishing live Irom dead cells include the loss oI
transport Iunction across plasma membrane which results Irom
loss oI membrane integrity.
Cells must be counted within 3-5 min because the number oI
blue-staining cells increases with time aIter addition oI the dye.
Large numbers oI samples have to be counted, it may be
inconvenient to perIorm all the tests on the same day by
counting one cell suspension at a time beIore staining the next
sample.
In engineered tissues, because oI the aIIinity oI the dye Ior
protein, trypan blue exclusion cannot be used to assess cell
viability and proliIeration in matrices
Exclusion dyes Exclusion dyes
1rypan blue
A stain which will only enter across the membranes oI
dead/non-viable cells.
- Cause cancer in lab. animals
- Appropriate precaution should be taken when handling trypan
blue (use oI extraction hood and gloves)
Dilution by trypan blue
- Viable cells : small, round and reIractive
- Non-viable cells : swollen, larger, dark blue
Hemocytometer
The most common routine method Ior cell counting which is eIIicient
and accurate is with the use oI a hemocytometer.
Hemocytometer Cell Counts Hemocytometer Cell Counts
Volume : 0.1mm
3
1 ml 1 cm
3
1000 mm
3
1 mm
1 mm
0.1 mm
Dead cell
Materials and Equipment
Trypan blue (0.4 g trypan blue in 100 ml
physiological saline) pass through a 0.22
Iilter
Hemocytometer with coverslip
Hand-held counter
Microscope
Methods
1) Clean hemocytometer & coverslip and wipe with 70 alcohol
beIore use
2) !lace coverslip on hemocytometer
3) Mix the cell suspension gently
4) Aliquot 0.1 ml cell suspensions
5) Add 0.1 ml (2-Iold dilution), 0.3 ml (4-Iold dilution) or 0.9 ml (10-
Iold dilution) trypan blue : appropriate range oI cells to be counted
6) Draw a sample into a !asteur pipette aIter mixing
7) Draw the cell suspension in to Iill the chamber
8) Using a light microscope at low power, count the number oI cells
9) Count the viable & non-viable cells in both halves oI the chamber
10) Calculations
A Vol. I cell solution (ml)
B Dilution Iactor in trypan blue
C Mean number oI unstained cells
D Mean number oI dead/stained cells
10
4
Conversion oI 0.1 mm
3
to ml
(1) Total number oI viable cells
A B C 10
4
(2) Total dead cell count
A B D 10
4
(3) To give a total cell count
Viable cell count dead cell count
(4) viability
(Viable cell count/Total cell count) 100
Example
Dilution Iactor
Vol. oI
CS
Cell count
Total viable cells
0.1 ml CS
0.1 ml TB (2)
20 ml 23
2022310
4
9.2 10
6
cells
0.1 ml CS
0.3 ml TB (4)
15 ml //
1542310
4
1.38 10
7
cells
0.1 ml CS
0.9 ml TB (10)
10 ml
//
10102310
4
2.3 10
7
cells
1) Vol. : Volume
2) CS : Cell Solution
3) TB : Trypan blue
Automated trypan blue method for
optimal cell viability determination
www.inno\atis.com
ntroduction
The trypan blue dye exclusion assay is the most commonly used
and accepted method for the measurement of cell viability
It relies on the alteration in membrane integrity as determined by
the uptake of dye by dead cells, thereby giving a direct measure of
cell viability
Based on optimal image analysis, the technology allow precise cell-
viability and cell-density determination
The system performs automatic and reproducible measurements
of human or animal suspension cell densities as well as
standardized differentiation between viable and dead cells, based
on the trypan blue dye exclusion method
The direct and automated optical analysis by means of modern
pattern recognition methods allows cell identification and a
standardized differentiation between viable and dead cells and also
cell debris
1he system consist of three functional part: the liquid handling
unit, image capture hard ware, and a data processing system,
including the user interface
Fig. Cedex workbench The IP Result viewer enables the user to control
whether the Cedex system recognizes the cells
correctly, and whether it reliably differentiates
and between viable and dead cells
Result view of the image processing
Marked viable and dead cells
Viable cell Dead cell
Fig. a) Distribution histogram of the viable cell diameter for a human leukemia
cell sample. b) Histogram of compactness for a human leukemia cell sample.
The abscissa represents the ratio of cell circumstance to cell area
normalized to the value of 1 for an ideal sphere
(a)
(b)
Ji Ji- -CE CE
1M 1M
CE Jiability Analyzer CE Jiability Analyzer
Principle
Trypan Blue dye Exclusion Methods
Run results
Electric Cell Counting using a
COU1ER

COUA1ER
ntroduction
Rapid, accurate, reproducible counts of cultivated cells can
be obtained using electronic cell-counting techniques
Used in range of cell counters and particle sizers
Used extensively in the biomedical field for routine
application and cell biology field for research tool
- blood cell counting, enumeration of immune complexes
and bacteriological investigation
Particles suspended in an electrolyte solution are drawn through
a small aperture or orifice in the wall of an electrical insulator,
across which a current path has been established by two
immersed electrodes
s each particle enters the aperture, it displaces its own volume
of electrolyte, thereby modulating the basic impedance of the
current
This is detected as a voltage pulse of short duration
Proportional in height to the volume of the displaced electrolyte
lso proportional to the magnitude of the particle size
The pulses produced by the
passage of cells through the
aperture can then be observed
by oscilloscope and analyzed
electrically to give a number
versus particle volume
distribution
lmost cells are spherical form and the results are expressed
conventionally as spherical equivalents` rather than by volume
By setting threshold limits to eliminate pulses being produced
by subcellular particles, and coincidence corrections for
doublets, triplets etc. of cells passing through aperture, the cell
concentration within any suspension can be determined
quickly and accurately
Tens of thousands of cells are counted, providing low statistical
deviations with an accuracy of measurement estimated by
Coulter to be within 1 of the true count, if the coincident
particle count is less than 15 of the total
Fig. Growth curve demonstrating the effect of various concentrations of EGF
on the growth of human ovarian adenocarcinoma cells
Calculation
The cell counts
obtained at each time
point may then be
graphically analyzed to
estimate the cell-doubling
time
ntroduction
Quantitative value Ior the loss oI cell viability
The activity oI LDH can be measured as the reduction oI pyruvate
to lactate.
The reduction is coupled to the oxidation oI NADH to NAD

,
which is Iollowed spectrophotometrically at 340nm
LDH
!yruvate NADH H

NAD

lactate
As NADH has a high absorbance at 340nm compared to NAD

,
the reaction is measured as the rate oI decrease in absorbance at
340nm.
DH (lactate dehydrogenase) eakage
Requirement of DH assay
Greater process productivity (e.g. High-cell-density entrapped-
culture systems) : diIIiculty oI cell isolation
Metabolic parameters(glucose uptake) : compromised because
uptake/production rates can alter as a result oI the cell switching
carbon source.
Analysis oI the release oI intracellular enzymes can be used
enzyme in cell culture studies is LDH.
Assumptions oI LDH assay
: Intracellular enzymes are only released aIter damage to the cell
membrane
: Rapidly released Irom damaged cells.
Pitfalls of DH assay
The release oI LDH activity can be related to the total No. oI dead &
lysed cells.
The stability oI LDH can vary considerably, ranging Irom the loss oI a
Iew percent per day to a halI-liIe oI 12h depending upon the cell type.
Assumed that the release oI LDH occurs rapidly aIter damage to the
cell membrane. This assumption is not necessarily correct.
The release oI LDH can be complete in cells which are considered
dead by dye exclusion methods.
Complete release may only occur upon cell lysis.
This point is Iurther complicated because dye exclusion methods do
not measure lysed cells.
Reagents and Solutions
BuIIer (Tris 81.3 mmol/L ; NaCl 203.3 mmol/L ; pH 7.2)
: Dissolve 4.92 g Tris and 5.95 g NaCl in 400 ml water and adjust
to pH 7.2 at 30with HCl. Make up to a Iinal volume oI 500 ml
with water
NADH solution(.-NADH 0.17 mg/ml)
: Dissolve 3.4 mg NADH in 20 ml buIIer
!yruvate solution(9.76 mmol/L)
: Dissolve 0.107 g monosodium pyruvate in 90 ml buIIer. Make
up to a Iinal volume oI 100 ml with buIIer.
BuIIer is stable at 0-4.
The NADH solution is kept at 0-4and must be prepared
Iresh daily
The pyruvate solution should be dispensed into 1.5ml
aliquots and stored at -20. AIter thawing, each aliquot
should be discarded.
The pyruvate solution is stable Ior 2 months.
Stability of solutions
Materials and Equipment
NADH solution
!yruvate solution
Narrow-bandwidth spectrophotometer, Iitted with a
thermostatted cuvette holder capable oI temperature control
within I0.1and a chart-recorder.
Assay Conditions
Incubation temperature : 30.0
Wavelength : 340 nm
Final reaction volume : 1.07 ml
Light path : 1.0 cm
Ethidium bromide (EtBr) and propidium iodide (!I)
!I binds to nucleic acids upon membrane damage : Ilow
cytometric techniques depend on Iluorescence, !I is
ideally suitable Ior the rapid evaluation oI the
permeability properties oI large numbers oI cells while
maintaining good statistical accuracy.
!I is impermeable to intact plasma membrane.
Intercalates with DNA or RNA red
Fluorescent dyes Fluorescent dyes
Fluorescein diacetate (FDA) is a nonpolar ester which passes through
plasma membranes and is hydrolyzed by intracellular esterases to
produce Iree Iluorescein, the polar Iluorescein is conIined within cells
which have an intact plasma membrane and can be observed under
appropriate excitation conditions.
Undamaged cell : highly Iluorescent Iluorescein dye
Damaged cell : Iluoresce only weakly
greenish-yellow at 450-480 nm
ntact cell -
P and FDA is added
Fluorescein in
intact cells
Schematic illustration of the principle of
PFDA cell viability assay
FDA {Fluorescein diacetate_
P {Propidium iodide_
Plasma membrane is damaged
; fluorescein leaks out
P enters and strains
nucleic acids
A group oI hepatoma cells exposed to a diIIusing wave oI digitonin. Intact cells
(green) are damaged by digitonin, loose the green Iluorescence and acquire red
Iluorescence oI !I.
Example 1 ; Observation of cell death
Functional assays Functional assays
Functional assays: evaluate viability by examining the
metabolic components that are necessary Ior cell growth, on
the premise that cellular damage will inevitably result in the
loss oI ability to maintain and provide energy Ior metabolic
Iunction and growth.
In terms oI tissue remodeling in implantation, speciIic protein
synthesis, such as collagen, by cells may be an important
Iactor Ior assessing the cellular Iunction related to cell viability.
The homeostasis oI Iibroblast collagen metabolism is regulated
in a complex manner by an interplay oI various diIIerent
mechanisms which include hormones, cytokines, and cell-
matrix interactions.
Colorimetric assay
Rapid and accurate assessment oI viable cell number
Miniaturized into 96-well plates
Measure using an Microplate reader
!ermit many sample to be analyzed rapidly
Reduce medium and plastics costs
These assay are read at 570 nm (except Ior the A! assay-
wavelength is 405 nm) on a ELISA plate reader, using a 620 nm
Iilter as reIerence wavelength.
It is important to remove any bubbles Irom the well beIore
absorbance readings.
MTT / XTT assay
Crystal violet dye elution (CVDE)
Acid phosphatase (A!) assay
Alamar blue oxidation-reduction assay
Neutral Red (NR) assay
|
3
H|-thymidine and BrdU incorporation
M11 Assay
Introduction
This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability, proliIeration
and activation oI cells.
The assay is based on the capacity oI mitochondrial
dehydrogenase enzymes in living cells to convert the
yellow water-soluble substrate 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a
dark blue Iormazan product which is insoluble in water.
The amount oI Iormazan produced is directly
proportional to the cell number in range oI cell lines.
metabolically active Cell
MTT
Formazan
Insoluble
Materials and equipment
MTT solution (5 /in phosphate buIIered saline (!BS) pH 7.5),
HCl, !ropan-2-ol
96-well microtiter plate, ELISA reader
Procedure (suspension and monolayer cells)
1. !repare MTT stock solution and Iiter through a 0.2 Iilter to
sterilize and remove the small amount oI insoluble residue
2. To 100 cell suspension or cell monolayer in each microtiter well
add 10 MTT
3. Incubate in a humidiIied incubator at 37Ior 3 h
4. Add 100 0.04 M HCl in propan-2-ol to each well and mix
thoroughly to dissolve insoluble dark blue Iormazan crystals
5. Read plate on a ELISA reader using a test wavelength oI 570 nm
and reIerence wavelength oI 630 nm
Procedure (immobilized cells)
1. !repare a solution oI MTT in !BS at a concentration oI 1 /. Add 1
MTT solution to 0.5 immobilized matrices (e.g. beads)
2. Incubate a 37Ior 3 h to allow MTT to diIIuse throughout the matrices
and react with cell
3. CentriIuge at 180 g Ior 2 min.
4. Measure optical absorbance at 570 nm. It may be convenient to dilute
Advantages of M11 assay
W Considered a major advance
; used the most prevalent in vitro assay
W Rapid, versatile, quantitative and highly reproducible
W Adaptable to large-scale screening
; relevant Ior most cells
W MTT reduction correlates to indices oI cellular protein
and earlier cell number
W More sensitive and earlier predictor oI toxicity than
classical LDH or neutral red measurements
Disadvantage of M11 assay
1. !roduction oI the MTT product is dependent on the
MTT concentration in the medium. The kinetics and
degree oI saturation are dependent on cell type.
2. Assay is less eIIective in the absence oI cell proliIeration.
3. MTT cannot distingulish between cytostatic and
cytocidal eIIect.
4. Individual cell numbers are not quantitated and results
are expressed as a percentage oI control absorbance.
5. Test is less eIIective iI cells have been cultured in the
same media that has supported growth Ior a Iew day,
which leads to underestimation oI control and untreated
samples.
Example; AH 12 313 cell
in medium
M11 solution
elution
bsorbance at 570 nm
11 assay
Introduction
The assay is based on the cleavage oI the yellow tetrazolium salt
XTT to Iorm an orange Iormazan dye metabolic active cells.
This conversion only occurs in viable cells.
The Iormazan dye Iormed is soluble in aqueus solution and is
directly quantiIied using Microplate reader.
Both MTT and XTT work by being to a Iormazan dye only by
metabolic active cells.
metabolically active cell
Water soluble
Materials and equipments
XTT labeling regent, electron-coupling reagent
96-well microtiter plate, Microplate reader
XTT labeling mixture : mixed 5 XTT labeling reagent
with 0.1 electron coupling reagent
Procedure
1. Cell are grown in microtiter plates in a Iinal volume oI 100
culture medium per well. The incubation period oI the
cell cultures depends on the particular experimental
approach and on the cell line.
2. AIter incubation period, add to each well 50 oI the XTT
labeling mixture
3. Incubate the microtiter plate Ior 4 to 24 h in incubator
4. Read plate on a Microplate reader using a wavelength
between 450 and 500 nm (reIerence wavelength oI 650 nm)
Compare with MTT assay and XTT assay
Culture cells in a MT!
Ior a certain period oI time (37)
MTT assay XTT assay
!repare labeling mixture
Incubate cells (0.5-4 h, 37)
Add solubilizing solution
(Isopropanol) and incubate
Measure absorbance using an ELISA reader
Add XTT labeling mixture Add MTT labeling reagent
Insoluble formazan Soluble formazan
Example: M11 and 11
MTT
XTT
Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002.
Evaluation oI redox indicators and the use oI digital scanners and spectrophotormeter Ior
quantiIication oI microbial growth in microplates. J. Micro. Methods. 50:63-73
!rinciple
Dye elution:
Cell up-taken dye was measured colorimetric method aIter acetic acid
dye elution.
Nuclei counting
Incubation oI cell samples in a mixture oI citric acid and crystal violet
causes cells to lyse and the released nuclei to stain purple.
Crystal violet
Procedure
Dye elution
' AIter removal oI medium, rinse 96
well plates with 100 /well oI
!BS and stain with 100 0.25
(g/10ml) aqueous crystal violet Ior
10 min.
_Rinse plats Iour times in tap water.
_ Dry the outsides oI the plates with
paper to help avoid water stains,
and then dry the plates at 37.
When dry, add 100 per well oI
33 glacial acetic acid (33
ml/100ml) and mix the contents oI
each well beIore reading at 570
nm.
Nuclei counting
' Allow microcarriers Irom a culture
sample (1ml) to settle to the
bottom oI a centriIuge tube.
_ Removed clear supernatant by
aspiration.
_ Add 1ml oI crystal violet reagent.
_ Incubate at 37at least 1 h.
Introduce a sample into the
hemocytometer chamber and count
the purple-stained nuclei as Ior
whole cells.
Example; Monolayer culture
ReI.:Journal oI rthopaedic Resarch19 (2001)
OP : Osteoporosis
Crystal Violet - 60minutes
Non-OP OP
Example; Microcarrier culture
Rabbit oral mucosal cell
cultured by microcarrier
Cell counting
Acid phosphatase (AP) assay
Introduction
The action oI this enzyme in many oI tissue is to cleave a
waste product called pyrophosphate and eIIectively
convert it to a useable phosphate.
!-nitrophenyl phosphate will be the substrate and
nitrophenol is the product oI this reaction.
Nitrophenol is colorless but when the pH oI the reaction
solution is alkaline, it is appears yellow. The pH oI the
reaction solution will be changed by the addition oI NaH.
!-nitrophenyl phosphate Acid phosphatase
Nitrophenol H!
4
-2
Materials and equipment
Substrate-containing buIIer : 10 mM !-nitrophenyl phosphate in
0.1 M sodium acetate pH 5.5, 1 M NaH
96-well micro titer plate, Microplate reader
Procedure
1. At end oI cell growth period, remove medium and rinse wells in
100 !BS
2. Add 100 substrate-containing buIIer to each well
3. Incubate Ior 2 h in incubator. Read plates at 405 nm, and either
reincubate Ior a Iurther time iI increased sensitivity is required, or
stop` with addition oI 50 /well oI 1 M NaH to cause an
electrophilic shiIt in the p-nitrophenol chromophore and thus
develop the yellow color, giving greatly increased sensitivity
!rinciple
In the presence oI cellular metabolism the color oI Alamar Blue (ALB) changes
Irom a Iully oxidized, non Iluorescent blue to a Iully reduced, Iluorescent red.
ALB will be reduced by a variety oI enzymes and small molecules, including
the cytochrome system, FMN, FAD, NAD, and NAD!.
Advantages
Simple, rapid, inexpensive, required no lysis, extraction or washing oI sample
Disadvantages
Unstable during storage (absorbance oI oxidized ALB-3day, reduced ALB-
increase Irom one day to the next)
Characteristics
- Sensitivity :
!ropidium iodide (!I), SulIorhodamine B (SRB) ~ ALBMTT
- The ALB assay is Iaster, simpler, and less arteIact prone than the MTT assay.
Alamar Blue oxidation-reduction assay
Procedure
' At the end oI an experimental incubation period, add 1 vol oI ALB stock solution per 25
vols (4v/v) oI growth medium in each well (8 ALB Ior 200 oI growth medium)
_ Incubate plates at 37Ior 3 h to allow metabolic dye reduction.
_ Equilibrate plates to room temperature Ior 30 min in the dark.
_ Measure the relative Iluorescence at 530~560 nm excitation and 590 nm emission
wavelengths.Fluorescence is temperature sensitive; either equilibrate plates in a warm
room at the culture incubation temperature. For better sensitivity, measure the Iluorescence
in bottom-reading rather than top reading mode. The ratio oI test to control Iluorescence
values at 590nm measures the eIIect oI a treatment on cell growth or metabolism.
For spectrophotometric assays, correct Ior the spectral overlap oI the oxidized and reduced
Iorms oI ALB by measuring each sample at two diIIerent wavelengths, between,
approximately, 540~630 nm . ne oI these must be a low wavelength (LW) and the other
a high wavelength (HW); Ior example, 570 ~ 600 nm, respectively.
A correct Iactor (R) Ior the absorbance oI oxidized alb must be calculated.
Measure the absorbance (AM) oI growth medium alone. (no ALB)
Measure the absorbances oI oxidized (blue) ALB in growth medium at the low and high
wavelengths.
Substract AM Irom each oI the measured ALB absorbance to produce, respectively,
ALW and AHW , the absorbance oI oxidized (blue) ALB at the low and high
wavelengths.
Calculate the correction Iactor R oI oxidized ALB:
RALW/AHW
Measure the absorbance values (ALW and AHW) oI a test sample at each
wavelength.
Calculate the percentage oI reduced ALB (ARLW) in a sample as:
ARLW 100 x |ALW-(AHW x R)|.
Calculate the percentage diIIerence in reduction (!DR) between treated and
control cells: !DR 100 x (test ARLW/ARLW Ior positive growth control)
Aeutral Red assay
(3-amino-7dimethyl-2-methyphenazine hydrochloride)
!rinciple
- The incorporaton oI NR into the lysosomes oI viable
cells aIter their incubation with test agents.
Use
- Industrial, pharmaceutical, environmental and other
testing laboratories concerned with acute toxicity
testing.
Advantages
- Simplicity, speed, economy, and sensitivity
Materials and Equipments
Solution
'Neutral red
4mg/ml stock solution
Dilute 1:100 into medium , incubate
overnight at 37and centriIuge Ior
10 min at 1500 g beIore use.
_ 1 CaCl
2
/0.5 Iormaldehyde
Mix 6.5 ml 37 Iormaldehyde with
50 ml 10 CaCl
2
and 445 ml distilled
water.
_ 1 acetic aicd/50 ethanol
Mix 4.75 ml acetic acid with 250 ml
95 ethanol and 245 ml distilled
water.
Equipment
'Complete media suitable Ior
chosen cell type.
_ Culture petri dish
_ 96well tissue culture plate
_ Inverted microscope
ELISA-type spectrophotometer
Microplate shaker
Eight-channel pipette
Procedure
' Resuspend cells oI actively growing culture and count cells and accurately allocate
appropriate number suspended in medium.
_ Seed 0.2 ml containing desired number oI cells to each well oI 96 well plate and incubate at
37Ior 24 h or longer.
_Removed the medium and add Iresh medium containing graded dilutions oI test agent.
Incubate Ior desired length oI time. Examine at least 4-8 wells per concentration oI test
agent.
Keep serum concentration as low as possible during this step.
(prevent or to reduce adsorption oI xenobiotic to serum components)
_AIter incubation Ior desired time interval, remove medium with test agent and incubate cells
with Iresh medium containing 40 /ml NR dye.
Continue incubation Ior 3h to allow Ior incorporation oI vital dye into survival cells.
Remove medium by inverting the plate and rapid rinse with a mixture oI 1 CaCl
2
/
0.5Iormaldehyde.
Extract dye into suprernate with 0.2 ml oI solution oI 1 acetic acid/50 ethanol.
AIter10 min at room temperature and rapid agitation Ior a Iew seconds on a micrometer plate
shaker, scan the plate with an ELISA-type spectrophotometer equipped 540 nm Iilter.
Example
ReI.:Biomaterials 21 (2000) 1549~1559
Fig. Monocellular cultures of "fibroblasts and
immortalized human gingival keratinocytes after
exposure to the materials (dye staining). Cell
derangement in the transition area of neutral
red destained - and stained cells, indicating
spindle- or fusiform cell shape in conventional
L-929 mouse fibroblasts (A) and rounded up
morphology in immortalized human gingival
keratinocytes (B) after methyl
methacrylate/monomer incubation.
Confluent cultures of L-929 cells (C) and
keratinocytes (D) exposed to Orthocryl Clear. n
both cultures, neutral red remains incorporated
in the membrane. Neutral red-fading in the
transition zone of an inhibition area after
Durabase exposure (E) and the cell damage
emphasized by trypan blue counterstaining (F).
Scale bars are 55 lm (A, C) and 90 lm (B, D, E,
F).
!rinciple
The rate oI DNA synthesis is a reIlection oI
proliIeration under many condition.To measure the
proliIerative rates by |
3
H|-thymidine uptake, cells are
cultured in microtitre wells, thymidine is added, and
the uptake by DNA is measured , aIter lysing and
washing on, by scintillation counting.
Bromodeoxyuridine(BrdU) can be incorporated
instead oI |
3
H|-thymidine and the incorporation can be
assayed with antibodies to BrdU in a non-radioactive
assay.

3
Hj-thymidine and BrdU incorporation
(DAA synthesis measurement)
Schematic diagram of
3
Hj-1dR and BrdU
abeling index with
3
Hj-thymidine
'Set up the culture at 2x10
4
cells/ml~ 5x10
4
cells/ml in 24 well plates
containing cover-slips. Grow to the desired cell density.
_Add |
3
H|-thymidine to the medium . 100KBq/ml(~5Ci/ml)and incubate Ior
the cultures 30 min.
_Remove the labeled medium, and discard it into a designed container Ior
radioactive waste.
_Wash the cover-slips three times with !BSA.
Add 1:1 !BSA: acetic methanol, 1ml per well, and remove it immediately
Add 1ml oI acetic methanol at 4 to each well, and leave the cultures Ior
10min.
remove the cover-slips, and dry them with a Ian
mount the cover-slip on a microscope slide with the cells uppermost.
Leave the mountant to dry overnight.
DAA synthesis by
3
Hj-thymidine
'Grow the culture to the desired density.
_ |
3
H|-TdR, 40 KBq /ml(~1.0Ci), 2 MBq/mol(~50 Ci/mol) in HBSS.
1Ci 3.7*10
10
/ s 3.7*10
10
Bq , 1Bq 1/s
_ Incubate the cell Ior 1-24 h.
_Remove the radioactive medium careIully.
Wash the cell careIully with 2 ml oI HBSS, !BSA, and 2 ml ice-cold 0.6 M TCA Ior 10
min.
Wash the cell with TCA twice 5 min each time.
0.5 ml oI 2 M perchloric acid, a hot plate at 60Ior 30min and allow the solution to cool.
Add 0.5ml SLS in NaH incubate the solution at 37Ior 30 min or overnight at room
temperature.
Collect the solubilized pellet and determine the radioactivity.
DAA labeling assay DAA labeling assay
(using fluorescent probes assay) (using fluorescent probes assay)
1wo types of nonviable cells
1wo methods:
Enzymatic DAA labeling and DAA-binding dye labeling
dNTP dUTP
Direct Indirect
X Fluoresein, FITC, PE etc Biotin DIG
vidin conjugated with
fluoresein, P, POD
nti-DIG antibody
conjugated with fluoresein,
P, POD
Enzymatic DAA labeling
Fig. Immunostaining of apoptotic cells (dark brown) by TUNEL and
peroxidase staining in rabbit endometrium
Fig. Flow cytometric histograms of control (left) and apoptosis-induced
cells (right) by PI and TUNEL labeling in HL-60 myeloid cells
DAA-binding dyes: fluorochrome
Dye
Permeability via
intact membrane
Staining
DN RN
cridine orange Yes Green Red-orange
1
Hoechst 33342 Yes Blue No
Hoechst 33258 Yes Blue No
DPI
(4,6-diamidino-2-
phenylindole)
Yes Bright blue No
EtBr
(Ethidium bromide)
No Orange Slightly red
1
PI
(Propidium iodide)
No Red No
2
1
RNase treatment is required
2
RNase treatment is required because PI could stain double strand RN
DAA-binding dye labeling
Dye
poptosis
Necrosis
Early apoptosis Late apoptosis`
cridine orange
Green
Condensed
Green
Fragmented
Green
Diffuse, Intact
Hoechst 33342
Blue
Condensed
Blue
Fragmented
Blue
Diffuse, Intact
Hoechst 33258
Blue
Condensed
Blue
Fragmented
Blue
Diffuse, Intact
DPI
Blue
Condensed
Blue
Fragmented
Blue
Diffuse, Intact
Ethidium bromide
No
(Orange, Condensed
if permeabilized)
Orange
Fragmented
Orange
Diffuse, Intact
Propidium iodide
No
(Red, Condensed
if permeabilized)
Red
Fragmented
Red
Diffuse, Intact
Pattern of dye staining according to color and
chromatin morphology
` late apoptosis is regarded as the stage of membrane fragmentation and secondary necrosis
Fig. In Hoechst 33258 / PI double staining, cells with blue intact
nuclei were viable cells, whereas those with blue fragmented nuclei
were early apoptotic cells. Cells with pink intact nuclei were
necrotic cells, whereas cells with pink fragmented nuclei were late
apoptotic cells. (blue against Hoechst33258, red against PI)
poptotic(0)
Necrotic(10.5)
poptotic(85.2)
Necrotic(11.2)
poptotic(1.2)
Necrotic(92.5)
Fig. DPI staining of condensed nuclei of apoptotic cells
Fluorescent protein biosensors measuring the molecular dynamics oI
macromolecules, metabolites, and ions in single cells have emerged
Irom the integrative use oI contemporary synthetic organic chemistry,
biochemistry, and molecular biology.
Vascular endothelial cells (ECs) play and important role in physiologic
hemostasis and blood vessel permeability, express immune related
Iunctions in monocytes and macrophages, and the viability oI ECs is
important in predicting the post-operative Iunction and durability oI
cryopreserved vessels Ior implantation.
Tetrameric Griffonia simplicifolia agglutins (GS1) shows prominent
binding only to the a-D-galactosyl residue oI blood vessel ECs.
Staining with Iluorescein isothiocyanate(FITC) conjugated with GS1
diIIerentiates ECs Irom the other cells in Ilow cytometry.
!I intercalates DNA double strands in dead cells
without regard to cell types, as their membrane lose
integrity.
GS1-FITC and !I double staining: presumed to
immediately determine the diIIerential viability oI
ECs Irom whole cells
The use oI Iluorescent probes enables the rapid
determination oI the viability oI ECs and whole cells
Irom the same tissue without separating ECs Irom
whole cells, and oI the viability oI each step in the
cryopreservation process
Fig. Each part oI quadrant statistics was observed under Iluorescence
microscopy. Live ECs preIerentially expressed the green color oI GS1. Dead
ECs are double stained by the green color oI GS1-FITC and the red color oI !I,
which results in yellow. Dead cells except dead ECs are identiIied by only the
red color oI !I.
Morphological assay Morphological assay
Large-scale, morphological changes that occur at the cell
surIace, or in the cytoskeleton, can be Iollowed and related
to cell viability.
Damage can be identiIied by large decreases in volume
secondary to losses in protein and intracellular ions oI due
to altered permeability to sodium or potassium.
Necrotic cells: nuclear swelling, chromatin Ilocculation,
loss oI nuclear basophilia
Apoptotic cells: cell shrinkage, nuclear condansation,
nuclear Iragmentation
Example; Morphological feature
(Human skin keratinocyte)
Fig. Morphological feature of () normal human skin
keratinocyte, and differentiated human skin keratinocyte(B).
() (B)
Example; Morphological feature
(Human skin fibroblasts)
Fig. Morphological feature of () normal human skin fibroblasts,
and aging human skin fibroblasts(B).
() (B)
Reproductive Assay Reproductive Assay
Clonogenic Cell:
DeIined as a cell with the capacity Ior sustained
proliIeration
Have undergone a minimum oI 5-6 doublings to give rise to
colonies containing at least 50 cells
Colony-forming Efficiency
Colony-forming Efficiency (CFE)
The ability to Iorm colonies is used as a measure oI
reproductive integrity
It is oIten reIerred to as plating eIIiciency (!E)
Aumber of colonies formed
CFE L 1"
Aumber of cell plates
Clonogenic assays
Be used to reIlect stem cell content
The basis oI assays Ior determining the lethal eIIects oI
cytotoxic agents
Determining the PE of an established
adherent cell line
Materials and Equipment
Cell growth medium : Eagle`s basal medium (BME)
100 iu/ml penicillin,
0.1 mg/ml streptomycin
Trypsin-EDTA
Gentain violet stain
Procedure
1. Trypsinize monolayer cultures or use cell suspension
cultures and determine the viable cell count
2. Dilute cells in growth medium to 1000 , 2000 and
5000 cells/10ml
3. Inoculate nine replicate !etri dished with 4 ml growth medium plus
1ml cell suspension
4. !lace plates in a humidiIied 5 C
2
plus air incubator are normal
growth temperature and rock shelI or tray gently to and Iro three
times. The plates must not be moved now until colonies are stained
5. Stain and count three replicate per cell density at 1,2 and 3 weeks
(murine lines) or 2 , 3 and 4 weeks (human lined)
6. Calculate the optimum cell densities Ior seeding and duration oI
incubation
Example; Rat keratinocytes
() (B)
(C) (D)
Colony forming Aon-colony
forming
48 br after
subculture
6 days after
subculture
: colony , : Single cells
aser Scanning Confocal Microscopy
; 3-D tissue viability assay
aser Scanning Confocal Microscopy
Laser scanning conIocal microscope (LSCM) is capable oI
observing selected thin layers oI a thick specimen placed under
a microscope. As a result, conIocal images have signiIicantly
less Iluorescence blur and out-oI-Iocus light and resolution as
well as contrast are improved.
This capability oI selectively observing thin layers oI a
specimen is called 'optical sectioning'. A series oI conIocal
sections can be combined into a three-dimensional image.
Combined use oI LSCM and Iluorescence provide powerIul
tools Ior intra-cellular phenomena and 3D tissue investigations.
Simplified Optics of aser Scanning
Confocal Microscopy
Principles of 3D image reconstruction
A series oI conIocal sections
A 3D image
Example of 3D image reconstruction
series of confocal sections
3D image
Example 2 ; 3-D viability of hepatoctye
spheroids
!at primary bepatocyte spbeorid `-D \iability ; ualitati\e
(quantitati\e assay is also possible using `-D image reconstruction|
Olympus LH-2 microscope
Epifluorescence linked witb a M!-5JJ confocal imaging system
lllumination by argon ion laser
Fluorescein diacetate(FDA|/Etbidium bromide(EL|
FDA ; clea\ed by esterase in cytoplasm of \iable cell
; fluoresces green
EL ; inserted in DNA of dead cells ; fluoresces orange
FDA/EL signals ; 51Jnm/56Jnm bandpass filter
\ariable plane of optical sectioning ; 1J15
Laser penetration dept ; 5J
Surface viability
3-D viability
Auclear Magnetic Resonance Methods
Monitoring oI cell metabolism
3-D observation oI high density perIusion bioreactor
ntroduction
Nuclear magnetic resonance (NMR), which was discovered in
1946, was used primarily by organic chemists Ior elucidation oI the
structure oI relatively small organic molecules.
NMR is now a proven technique Ior monitoring metabolism in
diverse systems-isolated cells and perIused organs, as well as the
intact animal and humans.
Studies oI cell metabolism have generally utillized the
31
!,
13
C,
1
H,
and
15
N nuclei.
Applications
Spin-echo NMR image can be used to monitor Iiber distribution
in the hollow Iiber bioreactor.
DiIIusion-weighted images can be used to map cell distribution.
Flow imaging can be used to map Ilow rates in both the Iibers
and the extracapillary space.
Chemical shiIt imaging can be used to map the distribution oI
cellular metabolites.
19F NMR imaging oI a perIluorocarbon probe molecule can be
used to map dissolved oxygen concentration.
Example ; Cellular energetics using
31
P AMR methods
1. Disslove cell extract in 5 mL oI
extract buIIer and remove
undissolved material by
centriIugation.
2. TransIer 3.5 mL oI the supernatant
to a 10 mm-diameter NMR tube
contatining 0.5 mL D
2
. In the
case oI
31
! NMR, MD!
contained in a coazial tube can
be used as a chemical shiIt and
quantitation standard.
3. Acquire NMR spctrum, maintaining
sample at a Iixed temperature,
typically 30. In the case oI
31
!
NMR, a 5 s interpulse delay and
a 60H Ilip angle pulse is
suIIicient to ensure complete
relaxation oI the metabolite
resonances.
Procedure
31
! NMR spectrum oI a perchloric acid extract oI
CH K1 cells growing in a hollow-Iiber bioreactor.
!ME: phosphomonoesters, !
i
: inorganic phosphate,
!DE: phosphodiesters, !Cr: phosphocreatine, AT!:
adenosin triphosphate, NAD

: nicotinamide adnine
dinucleotide, D!DE: diphosphodiesters.
Example ; Cell distribution imaging
Transaxial T
2
-weighted MRIs of coaxial hollow-fiber bioreactor for hepatocyte distribution
examination. Nearly void (B) and full of hepatocytes (C).
Example ; Fluid velocity distribution
imaging
Transaxial flow sensitive MRIs of a small (, intermal diameter 1.32 cm) and
a scaled-up bioreator (B, internal diameter 2.2 cm). The fluid velocity ranged
from zero (black) to around 2 cm/s (white).
Reference
1. Cell quantiIication, module 4B:1, Hemocytometer cell counts and viability
studies, 1.1 1.5
2. http://www.embl-heidelberg.de/ ExternalInIo/karsenti/countingcells. html
3. http://www.bd.com/clinical/!L/products/equipment/hemacytm.asp
4. !ark JC, Hwang YS, and Suh H., Viability Evaluation oI engineered
Tissue., onsei Medical Journal 2000 41(6): 836-844
5. Ashish A., Sonia SY, Mark AH, and Minas TC., !ressure Related
apoptosis in Neuronal Cel Lines., Journal of Neuroscience Research
2000 60: 495-503
6. Islam TC, Skarin T, Sumitran S, and ToItgard R., Retinoids induce
apoptosis in cultured keratinocytes., British Journal of Dermatology
2000 143:1709-719
7. Maria L. Anthony, Shane N. . Williams, and Kevin M. Brindle, Nuclear
magnetic resonance methods oI monitoring cell metabilism., Methods in
Biotechnology, Vol. 8: Animal Cell Biotechnology, 165-175, Edited by: N.
Jenkins, Humana !ress Inc. Totowa, NJ. 1999.
8. Macdonald JM, Grillo M, Schmidlin , Tajiri DT, James TL., NMR
spectroscopy and MRI investigation oI a potential bioartiIicial liver., NMR
Biomed. 1998 Apr; 11(2):55-66.
9. Flendrig LM, la Soe JW, Jorning GG, Steenbeek A, Karlsen T, Bovee
WM, Ladiges NC, te Velde AA, Chamuleau RA., In vitro evaluation oI a
novel bioreactor based on an integral oxygenator and a spirally wound
nonwoven polyester matrix Ior hepatocyte culture as small aggregates., J
Hepatol. 1997 Jun;26(6):1379-92.
10. Fluorescence and conIocal microscopy
(http://helios.mol.uj.edu.pl/conIc/main.htm)
11. Breuls RG, Mol A, !etterson R, omens CW, Baaijens F!, Bouten CV.,
Monitoring local cell viability in engineered tissues: a Iast, quantitative,
and nondestructive approach., Tissue Eng. 2003 Apr;9(2):269-81.
12. GriIIiths JB, Newell DG, and Doyle A., Cell & Tissue Culture: Laboratory
procedures., Cell QuantiIication, 4B, JHN WILEY & SNS Ltd,
UK.1993