MCL-5 Metabo-Tox Assay Kit FAQs
Below are the answers to many frequently asked questions submitted by
our customers. If there are other questions which you would like to have
answered please email us.
- What is included/not included in the Assay kit?
- What are the MCL-5 and cH2 cell lines?
- How many compounds can be tested with one BD Gentest™
MCL-5 Metabo-tox kit?
- How can both cell lines require the same selection markers?
- Can we order individual components from the kits as individual
items?
- What is the recommended dose range for a test compound
in the assay?
- How will common solvents such as DMSO potentially affect
the assay?
- How can I differentiate between direct and indirect cyto-toxicity?
- What will happen if the metabolites are less toxic than
the parent compound?
- How is it best to analyze the data?
- How important is obtaining cell counts and determining
doubling times for my assay?
- The cH2 cells appear to have a higher fluorescent signal
in the OBS plate than the MCL5 cells. Is this a problem?
- Can I take all my plates out to read on the fluorometer
at the same time?
- My data show only a 2 to 4-fold increase in NRFU from
my background control? Is this a sufficient signal?
- I am not getting a lot of signal with the BD Oxygen
Biosensor Systems. Is there something wrong?
- In some of my studies, the signal is higher for cells
with test compound than it is with cells alone (no compound). Why is
this happening?
- The wells containing media plus test compound (no cells)
have a higher signal the media only wells. What does this indicate?
- The fluorescent signal drops off dramatically to baseline
values, even in the lowest dose compound. What does this indicate?
- The fluorescent signal does not reach the base line
value. Can I calculate the IC50?
- Why use the optional BSO pre-treatment?
- Most viability assays require the subtraction of background
fluorescence. Why do I divide instead of subtract?
- Can we use the cells provided in the kit for other assays?
- Is it important to use both cell types in the assay?
Q: What is included/not included in the Assay kit?
A: We provide all the components
needed to conduct a study using five 96-well plates. The kit contains
4 x 110mL bottles of MediaPlus which contains all nutrient and
selection components required for the cell lines. The kit includes a vial
of both cell lines, the MCL-5 and the cH2 cells, Buthionine
Sulfoximine with which to inhibit GSH production and acetaminophen
as a control test compound. Five 96-well BD Oxygen Biosensor systems
are sent separately.
We do not include more toxic control substance such as NNK or dibenzopyrene
but the instruction manual contains information on how to obtain these
chemicals.
Q: What are the MCL-5 and cH2 cell lines?
A: MCL-5 and its control cell line,
cH2 are derived from AHH-1 TK+/- parental line, which is a human B lymphoblastoid
cell line derived from the RPMI 1788 cell line. A derivative of this cell
line L3, was found to have a 2 to 3 fold higher CYP1A1 activity. The AHH-1
TK+/- and L3 derivative were transfected with two different vectors both
of which contain the EBV origin of replication allowing them to stably
replicate as an extra-chromosomal plasmid.
To create the MCL-5 cell line, the L3 derivative of AHH-1TK+/- was transfected
with the pMF6/pMSR vector carrying cDNAs encoding CYP1A2, CYP2A6 and microsomal
epoxide hydrolase. In addition, cDNAs encoding CYP 3A4 and CYP2E1 were
introduced using the pEBVHistk/pHSR vector. The final MCL-5 cell line
expresses 5 xenobiotic metabolizing enzymes (CYP1A1, CYP1A2, CYP2A6, CYP
3A4, CYP2E1 and microsomal epoxide hydrolase). The cH2 cell line was created
using the parent cell line AHH-1 TK+/- in which empty vectors of pmF6/pMSR
and pEBVHistk/pHSR were transfected.
For more detailed information, refer to Crespi, CL et al (1990) The
development of a panel of human cell lines expressing specific human cytochrome
P450 cDNAs. In: ML Mendelsohn and RJ Albertini (eds) Progress in Clinical
and Biological Research, Vol 340B, Wiley-Liss, NY pp 97-106 and Crespi,
CL et al (1991) A metabolically competent human cell line expressing five
cDNAs encoding procarcinogen-activating enzymes: application to mutagenicity
testing. Chem. Res. in Tox. 4:565-572.
Q: How many compounds can be tested with one
BD Gentest™ MCL-5 Metabo-tox kit?
A: If the user wants to maximize
the screen, he/she can reduce the number of compound concentrations used.
So if a compound is tested with only 2 dilutions, then a total of 40 compound
could be tested. However, the smaller the number of compound dilutions
the less accurate will be the IC50 generated, if at all.
We have included two sample plate maps, both of which will allow the
calculation of IC50 using the BD Gentest™ MPM/ADMET
software program. The first allows for a 10-fold dilution series of
4 plus a blank. This allows 20 compounds to be tested in both cell types.
We have also included a method for a more thorough investigation of IC50
with a 3-fold dilution series of 9 concentration plus a blank. This would
allow 10 compounds to be screened in both cell types.
Q: How can both cell lines require the same selection
markers?
A: Both cell lines were transfected
with the same vectors except that in the case of cH2, the vectors were
empty. The two vectors, designated pMF6/pMSR (Davies et al., 1989. Carcinogenesis
11:885-891) and pEBVHistk/pHSR (Crespi et al., 1990 Carcinogenesis 11:
1293-1300) are selected by hygromycin B and by 2mM histidinol, respectively.
Both selection factors are already contained in the media.
Q: Can we order individual components from the kits
as individual items?
A: Extra components are available
as non-routine catalog items. Please contact Info_Gentest@BD.com for more
details.
Q: What is the recommended dose range for a
test compound in the assay?
A: For an unknown compound, biological
activity can range from picomolar to millimolar ranges; thus it is difficult
to provide an exact range to test for new compounds. Starting concentrations
will depend on compound solubility and the compounds' initial concentration
in DMSO. It is best to maintain the concentration of DMSO at <0.2%.
Q: How will common solvents such as DMSO potentially
affect the assay?
A: Organic solvents used to dissolve
substrates can inhibit cytochrome P450 enzyme activity [Chauret et al.
(1998) Drug Metab. Dispos. 26: 1-4 & Busby et al., (1999) Drug Metabo.
Dispos. 27: 246-249.] If solvents other than media or water must be used,
it is best to keep the concentrations of solvent as low as possible. Solvents
such as acetonitrile have the lowest P450 inhibition potential of several
common organic solvents and is compatible with the Oxygen Biosensor System.
DMSO which is commonly used, has a greater potential for P450 inhibition
and it is recommended that its concentration be <0.2%. Organic solvent
concentration should be controlled and normalized in each experiment.
For more detailed information on the effects of organic solvents on enzyme
activity see:
Appendix
A Effects of Solvents on the High Throughput Assay.
Q: How can I differentiate between direct and
indirect cyto-toxicity?
A: It is important to be able to
differentiate between toxicity of the parent compound (direct) and the
toxicity of a metabolite (indirect). In this assay, only the MCL-5 cells
carry the P450 enzymes thus if a metabolite is toxic, only the MCL-5 cells
will respond. In other words, the IC50 calculated for MCL-5 will be lower
then the IC50 calculated for cH2 cells. If the parent compound is toxic,
than both cells types will respond similarly or have the same calculated
IC50.
Q: What will happen if the metabolites are
less toxic than the parent compound?
A: We have not tested a full panel
of compounds in which the parent is toxic but the metabolites are not,
however, we have run across a couple that appear to behave in this fashion.
The compound terfenadine for example, was found to be less toxic to MCL-5
cells than to cH2 cells. This supports the existing data that terfenadine,
a compound known to have cardiotoxicity, is metabolized by CYP3A4 to its
relatively non-toxic metabolite, fexofenadine.
Q: How is it best to analyze the data?
A: There are three parts to analyzing
the data obtained by the BD Oxygen Biosensor system. This is made easier
for you with the BD Gentest™ MPM/ADMET
software program, which can normalize, analyze and store data obtained
from fluorescent plate readers.
Data obtained using the BD Oxygen Biosensor system first need to have
the background fluorescence units (FU) of the plate divided out. Please
note that in this instance background is divided and not subtracted. This
allows the user to directly infer the data as fold increase over background.
Each well is divided by itself with the fluorescent read obtained at the
"T=0" or immediately before plating the cells. (The t=0 values should
all equal 1 and values above that represent the growth of the cells).
The units are now referred to as Normalized Relative Fluorescent Units
(NRFU). For more detail, refer either to the MCL-5 Metabo-tox manual or
the FAQ's
for the BD Oxygen Biosensor System.(PDF file)
Data can be presented either as "percent maximal" or "percent inhibition".
- [NRFU (cells, no compound control)- NRFU (background, no cells)]
- Percent Maximal: Divide the NRFU of the test sample by the NRFU or
(Cells, no compound control) and multiply by 100. e.g. (0.45/1.9) x
100= 23.7% of Maximal signal
- Percent Inhibition: Subtract the percent maximal from 100. e.g. 100-23.7=
76.3% inhibition
The IC50 can be calculated using a number of different methods. We included
in the MCL-5 Metabo-tox Instruction manual a method for calculating
IC50 by linear interpolation.
For more information, see Addendum
III in Instruction manual).
The BD Gentest™ MPM/ADMET software program can be formatted to perform
the above functions automatically and then to calculate the IC50 using
either the four-parameter logistic fit or non-parametric smoothing spline.
For more information on the BD Gentest™ MPM/ADMET software program,
see MPM/ADMET
page
Our studies have shown that for much of the data generated, using the
non-parametric smoothing spline provided a much more accurate IC50 for
those data that do not conform to the criteria required of the 4-parameter
logistic fit. For more information on the spline method of calculating
IC50 see poster.
Q: How important is obtaining cell counts and determining
doubling times for my assay?
A: This is critical for two reasons.
One, the cell doubling times will provide the information by which to
assess the general health of the cells prior to starting your experiment.
The doubling time should be between 22 to 30 hours. Results from cells
with a very slow doubling time may give spurious results. The doubling
time may be longer immediately after thawing. If the times do not improve
with time, contact your BD Gentest Technical Specialist for more information
at info_gentest@bd.com.
Second, when the BD Oxygen Biosensor systems are being plated, it is
important to ensure that cells are sufficiently dispersed and counted
so that the initial seeding density is the same across the plate. Data
interpretation relies on this assumption. For calculation of cell doubling
times, see: (provide line to Addendum
III in instruction manual)
Q: The cH2 cells appear to have a higher fluorescent
signal in the OBS plate than the MCL5 cells. Is this a problem?
A: No. The cH2 cells have a slightly
faster doubling time then the MCL5 cells and because of the method we
use to analyze the data, it will not effect the overall interpretation
of the data.
Q: Can I take all my plates out to read on the fluorometer
at the same time?
A: To accommodate for high throughput
processing, multiple plates can be moved to the plate reader at one time.
It is best, however, to read the plates in the same order so that they
will have a roughly consistent temperature change. We do advise against
traveling a long distance with your plates as this does create airflow
across the plate and may decrease the over all signal. This should not,
however, undermine the accuracy of the calculated IC50. For optimal results,
the plates should be read one at a time straight from the incubator and
into a fluorometer that has been pre-warmed to 37oC. We recognize that
this is impractical except in situations where automation allows the direct
removal of plates from the 37°C incubator and into the plate reader.
Q: My data show only a 2 to 4-fold increase
in NRFU from my background control? Is this a sufficient signal?
A: Yes, even a 2-fold signal is sufficient
to detect dose dependent changes in fluorescence due to toxicity and to
accurately calculate an IC50. For more information, refer to Wodnicka
et. al. (2000) J. Biomol. Screening 5: 141-151.
Q: I am not getting a lot of signal with the
BD Oxygen Biosensor Systems. Is there something wrong?
A: There are a number of factors
that may impact the signal that you are getting. Make sure that the plate
reader that you are using excites and read samples from the bottom. The
signal from a top reading fluorometer is very much diminished. We have
used the following plate readers with excellent results: BMG Fluostar
or Polarstar, Cytofluor, Tecan and PE HTS 7000.
You can optimize the signal of your plate reader (e.g. the gain setting)
by following the optimization process outlined at the following links.
BD
Oxygen Biosensor Systems
Addendum VI of Instruction
Manual
Make sure that you are using the appropriate T=0 to blank your plates
and that you are following the protocol outlined in question 10.
Q: In some of my studies, the signal is higher for
cells with test compound than it is with cells alone (no compound). Why
is this happening?
A: We have often witnessed this,
as have others in the field. It is thought that this is due to a pre-toxic
increase in oxygen consumption before cell death ensues. When the data
are normalized to percent inhibition, these numbers will be negative meaning
no toxicity is occurring. As long as there are concentrations of compound
that bracket the 50% toxicity level, an IC50 can be accurately obtained.
In rare instances, an increase in signal could be due to auto-fluorescence
of the compound or of a particular metabolite. See question18 for more
details.
Q: The wells containing media plus test compound
(no cells) have a higher signal the media only wells. What does this indicate?
A: This indicates that the compound
is auto-fluorescent at the excitation and emission wavelengths required
for reading the BD Oxygen Biosensor System. The protocols outlined in
the instruction manual have measured only the highest concentration of
compound. This signal averaged with the one derived from media alone is
subtracted from the test wells prior to the calculation of the IC50. This
method will give an average background and is acceptable when there is
little to no background auto-fluorescence of the test compound. When there
is significant compound auto-fluorescence, it is necessary to provide
controls for each concentration of test compound so that each dose can
be specifically subtracted from the test wells. Again, this is done prior
to calculation of percent inhibition and IC50.
Q: The fluorescent signal drops off dramatically
to baseline values, even in the lowest dose compound. What does this indicate?
A: This suggests, provided that the
cells in absence of test compound have grown and have adequate fluorescent
signal, that the test compound is very toxic even at the lowest dose.
The dose range would need to be adjusted to the appropriate range to calculate
IC50.
Q: The fluorescent signal does not reach the
base line value. Can I calculate the IC50?
A: Calculating an IC50 from a curve
that does not reach baseline will depend on the curve generated. If the
curve does not reach 50% toxicity than an IC50 can not be calculated although
percent toxicity can. If the curve plateaus at a point lower than 50%
toxicity but higher than baseline than an IC50 can be calculated using
the non-parametric spline fit provided in the BD Gentest™ MPM/ADMET software
program. The four-parameter logistic fit would give inaccurate results
with this type of data. For more information see poster.
Q: Why use the optional BSO pre-treatment?
A: BSO inhibits the production of
glutathione (GSH), a protective agent in the cell. It can modify potentially
toxic metabolites, rendering them non-toxic. There have been drugs such
as acetaminophen that were released on to the market only to find hidden
hepatotoxicity issues due to GSH depletion in certain individuals. The
addition of BSO to this cellular assay does appear to impact those compounds
that are directly toxic or whose toxic metabolites are not modified by
GSH but does help identify those compounds that may have a hidden toxicity.
The pre-treatment chosen, 50uM for 3days, allows the pre-treatment to
go over a weekend and while large enough to deplete the GSH from the cells,
does not appear to have a negative impact on the cell. Over time and with
higher doses, the BSO will be toxic to the cells. Cells treated with BSO
should be treated with care. We do not think that BSO should interfere
with the metabolism of a particular test compound, as BSO is not metabolized
by the P450's.
Q: Most viability assays require the subtraction
of background fluorescence. Why do I divide instead of subtract?
A: Actually, the protocol asks that
you divide and subtract. There are two separate fluorescent issues in
this assay. The Oxygen Biosensor fluoresces in direct response to oxygen
present in the media or atmosphere. The signal due to ambient oxygen levels
in the well is an actual measure of oxygen present, not simply background.
Dividing out the ambient signal is the most statistically correct way
of normalizing the data.
Q: Can we use the cells provided in the kit
for other assays?
A: No. The label license included
with the kit restricts the use of the MCL-5 and cH2 cell lines to the
MCL-5 Metabo-Tox. If the cells are desired for uses other than Metabo-tox
Assay, the cell lines may be licensed separately. Contact info_gentest@bd.com
for details.
Q: Is it important to use both cell types in the
assay?
A: To differentiate between parent
compound toxicity and metabolite toxicity, both cell lines would have
to be used in the assay. If the MCL-5 cells were used alone, both types
of toxicity would be picked up but it would be difficult to determine
whether the response was from the parent or a metabo-toxin. 24. When a
difference in toxicity is observed between the two cell types, how certain
is it that this is due to a toxic metabolite? If a compound is found to
have a lower IC50 in the MCL-5 cells versus the cH2 cells and the difference
between the IC50 is greater than 2 -fold (at the very minimum), one can
be reasonably sure that there is a metabolic component. The greater the
fold-difference between the two numbers increases the certainty. As in
all experiments, the experiment should be repeated more than once and
the data confirmed.
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