F E A T U R E S
MICROCAT: A Novel Cell
Proliferation and Cytotoxicity
Assay Based on WST-1
ANN-MICHELE FRANCOEUR, Ph. D. and ALFRED ASSALIAN, M.D., F.R.C.S.C.
Department of Ophthalmology, Hôpital Notre-Dame
Montréal, Québec, Canada H2L 4M1
email: 104446.3321@Compuserve.com
Abstract
The MICROCAT (96-well microtest plate Consecutive Assay Testing) research
method was developed to accurately measure drug effects in vitro with small numbers of
cells (103-104 cells/culture). Standardization of MICROCAT using rabbit surgically
explanted primary eye cells (Tenon’s capsule fibroblasts, RTCF) is described. Results are
shown for TCF treated with mitomycin C, rapamycin, and c-myc antisense oligonucleotides. The principles of non-destructive testing were employed; a series of three
viable procedures were performed sequentially and repeatedly (i.e., daily) on the same
microculture without harming the cells. The first test performed was the WST-1 colorimetric test, which measures cell metabolism (dehydrogenase activities). This was
followed by Trypan Blue dye exclusion test for cell viability (cell membrane activity)
and then by photographic recording to obtain information on cell number, size, and
morphology. An optional final procedure was histological staining with a modified
Wright’s Stain prior to photographic recording. MICROCAT improved the accuracy of
our experimental results by minimizing the effect of inter-culture variations, which
might otherwise mask important drug effects. Other advantages include efficient use of
sample material, reduced drug testing costs, relatively rapid results, and increased
validity (through the use of normal cells derived from relevant tissue).
Methods
Cell culture
Primary RTCF were surgically explanted and cultured in monolayers in 96-well
flat-bottom microtest plates (Falcon #3072, Becton Dickinson), and used in passage
2– 6 as previously described (1). Cells were grown in 200 µl/culture media (Dulbecco’s
Modified Eagle Medium [DMEM] supplemented with 15% fetal calf serum [FCS],
2 mM glutamine, 100 µl/ml penicillin, and 100 µg/ml streptomycin). Cells were counted
and their viability assessed using Trypan Blue staining and hemocytometers (see below).
Changing cell culture media was effected by vacuum aspiration, taking care not to allow
the cultures to dry out. Multichannel pipettes and reagent reservoirs were used throughout MICROCAT, avoiding the introduction of air bubbles.
Drugs
Mitomycin C (MMC; Bristol-Myers Squibb Canada, Inc.), rapamycin (RAPA; ref. 1),
both at 100 ng/ml, and c-myc antisense phosphorothioate oligonucleotides (AS; 20 µm,
AAC GTT GAG GGG CAT, prepared by Laboratoire de Cardiologie Moléculaire, Hôpital
Notre-Dame) were diluted in media, and the cultures were incubated with these
reagents for the indicated times.
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CONTENTS
19
F E A T U R E S
WST-1 assay
The WST-1 assay from Boehringer
Mannheim was performed according to
the manufacturer’s specifications. It is a
colorimetric assay based on the cleavage
of WST-1 tetrazolium salt by mitochondrial dehydrogenases (part of the respiratory chain) and is a measure of cellular
respiration and metabolic rate. WST-1
reagent is light sensitive, and cell metabolism is temperature sensitive, so care
was taken to reduce light exposure and
maintain 37°C throughout.
The yellow-orange colored product, a
stable soluble formazan, was quantified
at 450 nm using an ELISA (microtest)
plate reader (model LP400, Diagnostics
Pasteur, Paris). This instrument facilitates
MICROCAT by simultaneously measuring absorbance of all 96 microtest plate
wells, thus allowing maintenance of cell
culture temperature, pH, and sterility.
Background absorbance was determined
by reading absorbance of cultures (cells +
media) at the start of the WST-1 assay
prior to reagent addition.
For purposes of standardization,
cultures received either 1, 10, or 20 µl of
stock WST-1 reagent in 100 µl of fresh
media (37°C). Cells were incubated for
1–4 hours at 37°C with duplicate hourly
absorbance measurements. Based on
these results, routine WST-1 assays were
performed for 1 hour with 10 µl reagent.
Cell viability assay
The classic Trypan Blue dye exclusion
staining method was employed. Cell
culture medium was removed after the
WST-1 assay by vacuum aspiration and
replaced with 50 µl of a 1:10 (v/v) mixture of 0.4% sterile-filtered Trypan Blue
Stain in Hanks Buffered Salt Solution
(HBSS). After a 2 minute incubation at
room temperature, the staining solution
was removed by aspiration and replaced
with 200 µl medium. The cultures were
viewed in an inverted microscope, and
the blue (dead) cells counted by eye
under 40X magnification. Alternatively,
the results were photographically
recorded (see below). Viability was
expressed as percentage unstained cells/
culture and required accurate total cell
count information.
Photographic recording
and data analysis
Cultures stained with Trypan Blue
stain or with Wright’s Stain (see below)
20
were photographed with a Nikon®
inverted photomicroscope or with a
Polaroid® Instant MicroCam camera.
Direct measurements of cell number,
size, and morphology were obtained
from prints, slides, negatives, photocopies, enlarged prints, or digitized
images thereof. Results were expressed as
percentage of control cultures or percentage inhibition relative to controls.
Modified Wright’s Stain
Cultures were fixed for 2 minutes with
100 µl of a cold (–20°C) 1:1 acetone:
methanol mixture and air dried for 30
minutes to overnight. Staining was performed with 50 µl of Wright’s Stain (diagnostic type R3375; BDH, Inc., Toronto) for
20 minutes and was followed by the addition of 50 µl of Wright’s pH 6.4 buffer
(BDH) directly on top of the stain for a further 10 minutes. The cell monolayers were
then washed twice by brief emersion in
phosphate-buffered saline (PBS), drained,
and air dried. Nuclei stained purple, and
cytoplasm stained pink.
Results
A MICROCAT experimental flow
chart is shown in Figure 1. The method
provides information on various cellular
parameters (cell metabolism, number,
viability, size, morphology, and number
of mitotic cells) by linking the new WST-1
colorimetric metabolic assay (2,3) to
classical cell biology procedures (dye
exclusion testing and photographic
recording). The nondestructive nature of
these procedures means that they can be
repeated in any order over experimental
times ranging from days to months.
Experiments may be terminated, and
cultures histologically stained with any
suitable method, such as the modified
Wright’s Stain described here.
The basic cell culture testing unit of
MICROCAT is shown in Figure 2 and
consists of replicate sets of cultures containing a range of cell concentrations.
Standardization of MICROCAT for a given
cell type (Figure 3) involves 4 steps.
Step 1, determining plating efficiency, is
needed for planning experimental time
courses, and this step is performed for
each type of cell to be studied. In contrast,
Steps 2– 4 involve calibration of different
assays, and a relevant cell type (e.g., rabbit
TCF) is appropriate for this purpose.
Typical results for standardization of
MICROCAT with RTCF cells are shown
in Figure 4 and Tables 1 and 2 (see also
“Materials and Methods”). In preliminary
experiments, growth curves were
performed with cultures seeded with
varying numbers of cells (250 – 4000
cells/well). The WST-1 assay was performed every other day with different
amounts of the WST-1 reagent and with
incubations from 1– 4 hours. Plating efficiency was poor in cultures seeded with
less than 500 cells (Figure 2). Maximum
absorbances in a 1 hour WST-1 assay
were observed with confluent healthy
cultures after 6–9 days in culture and
ranged from OD450 = 0.5 to OD450 = 2.8,
depending on the amount of WST-1
reagent used (Table 1A). Based on these
results, we chose the conditions for
PLATE CELLS IN SETS
(CONTROLS & TREATED)
CELL
METABOLISM
WST-1
ASSAY
ELISA PLATE
READER
ABSORBANCE
DATA
DATA
ANALYSIS
CELL
VIABILITY
CELL NUMBER
SIZE, MITOSES
MORPHOLOGY
TRYPAN BLUE
VIABILITY
STAIN
WRIGHT'S
STAIN
MICROSCOPE
PHOTGRAPHIC
RECORDS
DIGITAL
IMAGE
Figure 1 MICROCAT flow chart. Schematic diagram of three consecutive assays performed in MICROCAT
(WST-1, Trypan Blue Stain, Wright‘s Stain). To the left is indicated the cell biology information obtained from the
assays, while to the right are listed the equipment required, the format of the results, and how they may be
processed and analyzed manually with an ELISA plate reader and a standard photomicroscope (or by eye).
Alternatively, microscopic images may be recorded with a digital camera and viewed directly via computer monitor.
BIOCHEMICA
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NO. 3 [1996]
CONTENTS
F E A T U R E S
2000
2500
1000
1500
250
Number of
cells plated
Replicate
Cultures (4)
500
1 SET OF MICROCULTURES
Post WST-1 Assay
Plate
identification
96-Well
Microtest
Plate
High-concentration
drug controls
DRUG A-Treated
Cultures
Drug B-Treated
Cultures
Figure 2 MICROCAT experimental unit. Schematic diagram of one set of microcultures and photograph of a
96-well microtest plate with two sets of microcultures taken 15 minutes after a 2 hour WST-1 assay. The sets
shown here are from Day 7 of an experiment in which RTCF cultures in Panel A received rapamycin (100 ng/ml)
and those in Panel B received mitomycin C (100 ng/ml) for 2 days, and then were allowed to recover for 5 days
without drug treatment. Included in this assay were high-concentration drug controls, which helped to verify that
the drugs were active.
As can be seen, the healthiest cultures are readily detected by eye because of their bright orange color, while cultures with poor plating efficiency (wells 1-4) or which experienced cytotoxicity due to high mitomycin C levels
(wells 5-7) can be easily detected by eye and are pink in color.
STEP 1
DETERMINE
CELL TYPE
PLATING EFFICIENCY
A) ELISA PLATE READER
1 -FILTERS
2 -ACCURACY
3 -PRECISION
4 -BACKGROUND
B) STATISTICS
1 -METHOD
2 -NO. OF
REPLICATE
CULTURES
REQUIRED
STEP 3
CALIBRATE
TRYPAN BLUE
STAINING
STEP 4
CALIBRATE
WRIGHT'S
STAIN
C) PHOTO-MICROSCOPE
MICROCAT RESULTS
200,0%
150,0%
% CONTROL
STEP 2
CALIBRATE
WST-1
ASSAY
CONTROL
DRUG A
100,0%
DRUG B
DRUG C
50,0%
0,0%
Metab. Cell No. Viability Mitosis Cell Size
ASSAY
Figure 3 MICROCAT calibration. The 4 steps of MICROCAT are illustrated together with idealized results
comparing metabolism, cell number, viability, mitosis, and cell size of treated cultures (Drugs A-C) vs. Controls.
Steps 2-3 can be performed with a “standard” cell type (such as rabbit TCF) and thus monitored for quality control. Step 1, determining plating efficiency, varies with different cell types (e.g., rabbit TCF cells from high vs. low
passage numbers) and influences experimental design.
BIOCHEMICA
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NO. 3 [1996]
CONTENTS
routine MICROCAT WST-1 assays as
10 µl of WST-1 reagent and 1 hour incubation time (conditions recommended
by the manufacturer, confirming that
they may be generally applicable for different cell types).
The routine MICROCAT WST-1 test
on subconfluent cultures typically yielded
maximum absorbances around 1.5 to
1.8 OD units before subtraction of background absorbance (Tables 1B and 3 and
Figure 4D). This allowed us to detect
drug stimulatory effects as well as
inhibitory effects, as the ELISA plate
reader was capable of absorbances as
high as 3 OD units at 450 nm (Figures
4A and 4B). Alternatively, 5 µl of WST-1
reagent was used in routine assays when
confluent cultures were employed.
Background values (culture dish
plastic + media + cells + drugs or media
additives) for MICROCAT were determined from absorbance readings taken at
450 nm just prior to the addition of
WST-1 reagent and then subtracted from
experimental values. Backgrounds were
similar in the presence or absence of the
WST-1 reagent and ranged from 0.16 to
0.21 OD units in cultures with low vs.
high cell densities (Table 1B and Figure
4D). Using other filters as a “reference
filter” was found to be less accurate, as
absorbance tended to be lower by 0.05 –
0.15 OD units (Table 1D). This would
lead to an underestimation of druginduced inhibition of cell metabolism,
especially in sparse cultures. Phenol red
indicator dye absorbs about 0.1 OD
unit/100 µl volume media at 450 nm
(Table 2); thus, sensitivity may be
improved by omitting the dye from the
culture media.
In our hands, RTCF needed 2–3 days
in culture to establish themselves, and
conditions of low plating efficiency could
readily be detected by eye (see for
example, Figure 2, Panel A) due to the
difference in color of healthy (orange) vs.
unhealthy (pink) cultures after the
WST-1 assay. To avoid complicating
experiments by poor plating efficiency,
RTCF were routinely plated three days
prior (Day -3) to addition of a drug on
Day 0, and time points were typically
taken at Day 0, 1, 3, and 7 (Figure 4D).
The WST-1 assay sensitivity limit for
RTCF cells was about 100 cells/well,
which gave an absorbance of 0.05 OD
units greater than background.
21
F E A T U R E S
3,2
2,8
2,8
2,4
2,4
OD 450 nm
1,6
1,2
OD 450 nm
1
2,0
C
D
1,6
1,6
0,8
0,4
0,4
0,2
,01
0,0
0
ul WST-1 Product
100
200
ul WST-1 Product
300
W150 ul E1
W150 ul E2
W 200 ul E1
W 250 ul E1
W 300 ul E1
300
BGD
0,0
W 50 ul E1
W 50 ul E2
W 100 ul E1
W 100 ul E2
200
500
0,6
0,8
100
1000
1,0
0,4
0
1250
1,2
2,0
0,8
0,0
1500
1,4
1,2
,1
CONTROL CULTURES
1,8
Plate
Med 100 ul
Med 200 ul
OD 450 nm
B
10
OD 450 nm
A
3,2
-1
0
1
2
3
4
5
6
7
Days Post Drugs
Figure 4
WST-1 assay calibration.
Panel A. Standard curve relating absorbance at 450 nm to increasing amounts of culture media post WST-1 assay (containing orange-colored product). This type of curve has
various uses, such as (i) facilitating experimental design (e.g., determining what culture media volume to use when relating one cell type to another or when scaling up from
one culture plate size to another) or (ii) for calibration of pipettes or ELISA plate readers or filters (see Table 2).
Panel B. Same standard curve data as in Panel A, depicted with a logarithmic Y axis and with error bars omitted for clarity, to illustrate the high sensitivity of the ELISA
plate reader.
Panel C. Comparison of various sources of experimental error in the WST-1 assay. Absorbance at 450 nm and experimental error (based on 4 replicates) of microtest plate
plastic (Plate), different volumes of media (Med), duplicate (E1 and E2) ELISA plate reader data for the same WST-1 product volumes (W).
Panel D. Representative WST-1 assay results for an experiment with RTCF (passage 6) where sets of microcultures were plated with 500 –1500 cells/culture (4 replicates) at
Day –3, and WST-1 assay time points were taken at Days 0, 1, 3, and 7. Media was changed on Days 0 and 4. For clarity, the data is shown without error bars, which varied
from ±3–22%. Background (BGD) was subtracted from experimental data and is also shown separately for comparison.
SD, standard deviation; SE, standard error; CV, coefficient of variation.
Sources of WST-1 experimental variability are listed in Table 2. Media
containing the orange colored product
(OD >1.2) was collected post WST-1
assay, pooled, frozen, and subsequently
used to calibrate instruments such as the
ELISA plate reader (see Figure 4C), filters
(Table 1C), and multichannel pipettes
(Table 2). While absorbance was proportional to volume of WST-1 (Figure 4A),
the precision of the plate reader varied
from a low of 2% to a high of about 8%,
with maximum imprecision between
1.4–2.2 OD units (Figures 4A, 4C, and
Table 2). Thus, the experimental variations introduced by the ELISA plate
reader were sometimes comparable to
inherent inter-culture variations (5 – 40%)
seen with primary cultures (cell strains);
see Figure 6 and Table 3 for examples.
Referring to Tables 1 – 3, by far the largest
sources of experimental variations were
the interculture variations, followed by
the ELISA plate reader and multichannel
pipettes. An avoidable large source of
experimental error was due to air bubbles
present during the ELISA plate absorbance measurements, which could increase
absorbance by 0.8 OD units.
We developed procedures to detect
and rule out outliers (defined as data
greater than 3 standard deviations from
the mean) in our data and to carefully
22
track errors in all experimental manipulations. Two procedures improved our
ability to detect outliers: (i) We continued the WST-1 incubation for a
second hour and compared the hour 2
data to the hour 1 data when an outlier
was suspected. (ii) Duplicate ELISA plate
readings, about 1 minute apart, were also
taken at each experimental time point
and after each hour of incubation (see
Figure 4C). Both procedures are now
routinely performed and have helped to
trace the source of the variation to inherent inter-culture variation, variation
introduced by the ELISA plate reader,
human error, or other equipment failure
(e.g., printer or pipetting error).
For microcultures with high absorbance values in the WST-1 assay (OD
>3.0), 10 µl and 50 µl aliquots were
removed and the absorbance reread at
450 nm. Results were then corrected for
volume using a standard curve (Figure
4A). This method is recommended by
Boehringer Mannheim and proved more
accurate than dilution of samples (data
not shown). Alternatively, cultures with
high absorbances could be reread with
other filters (wavelengths of 405 nm or
490 nm), and the results could be multiplied by the empirically derived
absorbance correction factor constants
indicated in Table 1C.
Calibration of MICROCAT step 3 was
straightforward. Trypan Blue staining was
performed as described in “Materials and
Methods.” Reversing the order of the WST-1
and Trypan Blue tests did not significantly affect the results of either test
(data not shown), at least for the short
incubation times used. Viability was
reduced in cultures that had been exposed
to either reagent for 4 hours (< 90%
viability) or overnight (< 50% viability).
MICROCAT step 4, the calibration of
Wright‘s Stain, was similarly straightforward (see “Materials and Methods”
and Figure 5).
MICROCAT offers the possibility of
research testing drug effects on small numbers of primary cells in vitro (Figure 6).
RTCF cultures were plated (Day -3),
grown for 3 days, and then treated with
mitomycin C (100 ng/ml), rapamycin
(100 ng/ml), or c-myc anti-senseoligonucleotides (20 µM) for 2 days, and
then allowed to recover for 5 days.
Figure 6 shows WST-1 data for the Day 3
time point (1250 cells plated on Day -3).
The statistical analysis of this data is provided in Table 3, while a summary of the
MICROCAT experiment is presented in
Figure 7 (error bars omitted for the sake
of clarity). Figure 5 displays representative photographic records used to count
cells, measure cell size, and assess
BIOCHEMICA
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NO. 3 [1996]
CONTENTS
F E A T U R E S
B
A
C
D
morphology of control and drug-treated
cultures for Day 7 of this experiment.
All three types of drugs produced a
similar 50– 60% inhibition of cell metabolism and blocked mitosis with relatively
little effect on cell viability (Figure 7).
The effects on cell number and size were
more complex and in some cases (c-myc
antisense or rapamycin) were reversible.
While mitomycin C (Figure 5, Panel B)
caused the cells to grow very large (2.5
times the area of control cells, Panel A),
rapamycin had little effect on cell size
(Panel C), and c-myc antisense-treated
samples contained very many tiny cells
(Panel D).
These results extend our previous
results with rapamycin (1) and support
observations made by other groups using
RTCF cells and mitomycin C (4–6). A
detailed report of our work with MICROCAT and human and rabbit TCF are
beyond the scope of this article and will
be presented elsewhere (Francoeur and
Assalian, manuscript in preparation).
Discussion
Figure 5 Photographic records of MICROCAT assays. Shown are Wright’s stained cultures on Day 7 (5 days
recovery after 2 days of treatment with drugs) from the same experiment described in the text and shown in
Figure 6.
Panel A: Control cultures
Panel B: Cultures treated with 100 ng/ml mitomycin C
Panel C: Cultures treated with 100 ng/ml rapamycin
Panel D: Cultures treated with 20 µM c-myc antisense
oligonucleotides.
MICROCAT is being used at the
research level to study the effects of various drugs currently used as glaucoma
adjuvant therapy (e.g., mitomycin C,
5-fluorouracil) and potentially useful
drugs (rapamycin and c-myc antisense
MICROCAT SUMMARY - DAY 3
(1500 Cells plated Day -3)
WST-1, RTCF, Day 3
(1250 Cells plated on Day -3)
1,8
1,6
120,0%
1,4
100,0%
1,0
0,8
0,6
0,4
0,2
0,0
CONTROL AS20
RAPA
MITC
BGD
TREATMENT
Figure 6 Histogram showing data from a representative MICROCAT WST-1 experiment with
RTCF cells. Cells were plated with 1250 cells/well
on Day -3, treated with the indicated drugs for 2
days (Day 0-Day 2), and allowed to recover for 5
days as described in the text. (See Table 3 for statistics).
AS20 = 20 µM c-myc antisense oligonucleotides;
RAPA = 100 ng/ml rapamycin; MITC = 100 ng/ml
mitomycin C.
BIOCHEMICA
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NO. 3 [1996]
CONTENTS
% OF CONTROL VALUES
OD 450 nm
1,2
CONTROL
AS20
RAPA
MITC
80,0%
60,0%
40,0%
20,0%
0,0%
Metab.
Cell No. Viability
ASSAY
Mitosis
Figure 7 Summary of MICROCAT DATA for Day 3 of the experiment described in the text and in part in
Figures 5 and 6. The results were expressed as percentage of control culture values for purposes of comparison.
23
F E A T U R E S
Volume of WST-1 (µl)
Maximum OD450
20
10
2
1
2.8
1.8
0.7
0.5
A) Standardization of WST-1 assay on RTCF cells. 4 replicates, 1 hr 37°C, 100 µl media; 6 days in culture,
4000 cells plated on Day 0.
OD450
Coefficient
of variation
Standard
deviation
Standard
error
Routine assay, maximum mean
(4) absorbance observed
at any time point
Day 6 in culture
= 1.488 ± 0.204 (13.7%)
11.0
0.163
0.058
Mean background absorbance
+ WST-1 reagent (cells + 100 µl
media + plastic); n = 16
Day 6 in culture
0.169 ± 0.012 (7.1%)
3.4
0.006
0.001
Mean background absorbance
– WST-1 reagent (cells + 100 µl
media + plastic); n = 16
Day 1 (min) – Day 6
(max) in culture
OD = 0.166 – 0 191
± (7.4%)
4.4
0.007
0.002
B) Routine WST-1 assay on RTCF cells. Absorbances at 450 nm were measured on confluent cultures
(1000 – 1500 cells plated Day -3); 100 µl media, 98% viable cells.
Absorbance correction factors
450 nm
405 nm
490 nm
x 1 (none)
x 1.25
x 3.22 (OD < 0.25)
x 2.63 (OD = 0.26 – 0.60)
C) Comparison of ELISA plate filters in WST-1 assay with RTCF cells.
Mean of 18 determinations
(cells plus 100 µl medium plus plastic)
450 nm
630 nm
0.193 ± 4%
0.124 ± 4%
D) Comparison of backgrounds in WST-1 assays.
Day 3 measurements, 1500 RTCF cells plated; 10 µl, 1 hr.
Table 1. Various measurements taken during standardization of WST-1 assay for MICROCAT.
WST-1 Assay: Sources of variation that influence
experimental error.
Absorbance at 450 nm
Differences between different ELISA plate readers
measuring the same sample (comparison of 96 samples)
0.05-0.15 OD units
± 5% – 15% (depends on OD)
Differences between different channels of a
multichannel pipette (8 or 12)
(calibration of 2 different pipettes)
0.05 – 0.10 OD units
± 5 – 13% (depends on OD)
Differences between means of 2 consecutive sets
of ELISA plate readings of a set of 8 samples of
100 µl WST-1 product of absorbance
OD = 1.00 (same machine)
<< 0.05 OD units, < 1%
mean OD = 1.007 + 0.038 (3.8%)
(both determinations gave the same mean)
Differences between absorbance of plastic of different
wells on microtest plate and between 2 different
microtest plates (96 determinations on each plate)
<< 0.05 OD units, < 1%
mean OD = 0.034 ± 0.003 (8.8%)
(for 1 or 2 two plates)
Differences between background absorbance of
cultures (plastic + cells + 100 µl media) with
different cell densities. Comparison between high
(Day 6) and low (Day 1) RTCF cultures.
< 0.05 OD units
0.170 ± 0.014 (8.2%) (low)
0.202 ± 0.031 (15.3%) (high)
Difference OD = 0.032 (8.8%)
Absorbance due to 100 µl media alone (includes plate
but no cells, mean of 18 different determinations,
same pipette used for each). No statistically significant
difference observed with or without WST-1 reagent.
<< 0.05 OD units
0.105 ± 0.007 (6.7%)
Differences due to presence of small or large air
bubbles in 100 µl media in wells of microtest plate,
includes plastic but no cells
+ 0.1 – 0.785
(can be much greater than sample absorbance)
oligonucleotides) in vitro, with small
numbers of patients’ early passage TCF
cells. Antisense oligonucleotides (reviewed
in ref. 7), particularly those that block the
expression of such key cell cycle control
proteins as c-myc, offer the promise of
new therapeutics in ophthalmology, with
high specificity and minimal side effects.
MICROCAT largely depends on the
sensitivity and reproducibility of the WST-1
assay and the fact that this test is not cytocidal, at least during short (1–2 hour)
assays. The Trypan Blue dye exclusion test
is useful for assessing membrane integrity/
cell viability, but is not informative when
cells have functioning membranes but low
or no potential for proliferation (as
occurred with mitomycin C).
We found the Polaroid MicroCAM
camera valuable in speeding up results
and expect that a digital camera would
facilitate experimental throughput even
more. The improved accuracy and validity
of our results with MICROCAT relative to
other methods of drug testing/evaluation
becomes more evident as the inherent
inter-culture variations become large
(>± 30%, CV >15).
This first communication on MICROCAT involves manual testing; however,
automation is certainly feasible, as are
further miniaturization and additional
types of testing (e.g., PCR-based testing).
Clearly, the MICROCAT can accommodate other types of cells, as well as other
drugs and variations in the sequence and
type of intermediate non-destructive testing performed. A limiting factor is our
inability to obtain 96-well microtest
plates for tissue culture with detachable
wells (as have been developed for
immunological assays) or with UV-permeable wells. This would allow us to continue microculture testing with a longer
series of tests using other techniques (e.g.,
immunochemical, molecular biological,
biochemical).
References
1. Salas-Prato, M., Assalian, A., Mehdi, A. Z., Duperré,
J., Thompson, P. and Brazeau, P. (1996) Inhibition by
rapamycin of PDGF- and bFGF-induced human
Tenon fibroblast proliferation in vitro. J. Glaucoma
5:54-59.
2. Ishiyama, M., Masanobu, S., Sasamoto, K.,
Mizoguchi, M. and He, P. (1993) A new sulfonated
tetrazolium salt that produces a highly watersoluble formazan dye. Chem. Pharm. Bull. 41:11181122.
Table 2. Sources of variation in WST-1 assay.
24
BIOCHEMICA
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NO. 3 [1996]
CONTENTS
F E A T U R E S
Statistic
Control
c-myc
antisense
oligonucleotide
(20 µM)
Rapamycin
(100 ng/ml)
Mitomycin C
(100 ng/ml)
Background
% of control
100%
50.0%
47.6%
36.2%
0.0%
% inhibition
0%
50%
52.4%
63.8%
–
Mean
background
1.359
0.679
0.647
0.492
0.000
Mean
1.539
0.859
0.827
0.595
0.180
Range
(min-max)
1.340 – 1.765
0.695 – 1.164
0.583 – 1.082
0.475 – 0.682
0.173 – 0.189
Range
0.425
0.469
0.499
0.207
0.016
Error
0.212 (13.8%)
0.234 (27.2%)
0.250 (30.2%)
0.103 (17.2%)
0.008 (4.4%)
Standard
deviation
0.145
0.149
0.187
0.065
0.005
3. Ishiyama, M., Tominaga, H., Shiga, M., Sasamoto, K.,
Ohkura, Y., Ueno, K. and Watanabe, M. (1995) Novel
cell proliferation and cytotoxicity assays using a
tetrazolium salt that produces a water-soluble formazan dye. In Vitro Toxicology 8:187-190.
4. Yamamoto, T., Varani, J., Soong, H. K. and Lichter, P.
R. (1990) Effect of 5-fluorouracil and mitomycin C
on cultured rabbit subconjunctival fibroblasts.
Ophthalmol. 97: 1204-1210.
5. Khaw, P. T., Ward, S., Porter, A., Grierson, I.,
Hitchings, R. A. and Rice, N. S. C. (1992) The longterm effects of 5-fluorouracil and sodium butyrate
on human Tenon‘s capsule fibroblasts. Invest.
Ophthalmol. Vis. Sci. 33: 2043-52.
6. Bershadskii, A. D. and Stavrovskaia, A. A. (1991) The
normalization of tumor cell morphology related to an
increase in their size: research on giant cells produced by mitomycin C treatment. Tsitologiia 33:6775 (title translated from Russian).
7. Prins, J., de Vries, E. G. E. and Mulder N. H. (1993)
Antisense of oligonucleotides and the inhibition of
oncogene expression. Clinical Oncology 5:245-252.
t-Test sig. C/Dx* 0.0
00
0.0
00
0.0
00
0.0
00
0.7
06
0.0
00
0.0
00
0.7
06
0.0
05
0.0
00
0.0
00
0.0
05
0.0
00
0.0
00
0.0
00
Significant*
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Coefficient of
variation
9.4
17.4
22.6
10.8
3.0
Acknowledgments
Standard error
0.051
0.053
0.066
0.023
0.002
Support for this study was provided
by the ophthalmologists of Hôpital
Notre-Dame and Alcon Canada, Inc.
Table 3. Statistical analysis of representative MICROCAT WST-1 experimental data illustrated in the histogram in Figure 6 and for the experiment described in the text. A representative MICROCAT WST-1 Day
3 time point (1250 cells plated on Day - 3) was assayed after 2 days of drug treatment (Days 0 – 2) and 1 day of
recovery.
*t-Test sig. C/Dx: Comparison of Student t-test significances of WST-1 results for control cultures vs. those of drug
X-treated cultures
BIOCHEMICA
■
NO. 3 [1996]
CONTENTS
Product
Cell Proliferation
Reagent WST-1
Cat. No.
1 644 807
Pack Size
2500 tests
25