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Reagents
and materials
Mynox®
Reagent
Sterile,
ready-to-use solution in phosphate-buffered saline (PBS)@ pH 7.4,
aliquoted per tube for single applications, 220
µl/tube.
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Catalog
number |
|
U110523 |
MYN-200 |
2
treatments |
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U110524 |
MYN-500 |
5
treatments |
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U110523 |
MYN-1000 |
10
treatments |
Stability
and Storage
Mynox®
is stable until the expiry date given in the Guarantee Certificate if
stored at +2 °C to +8 °C.
Supplemental
Requirements
Standard
cell culture equipment
Cell
culture medium, fetal calf serum, phosphate-buffered saline (PBS)
Sterile
plastic ware
Mycoplasma
detection system to verify the elimination success, e.g. Minerva
Biolabs’s VenorGeM Mycoplasma PCR Detection Kit
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Introduction
For
both biological and economical reasons, it is important to eliminate
mycoplasma from cell cultures being used for basic research, diagnosis,
and biotechnological production. The most commonly used method for
elimination, inactivation, or suppression of mycoplasma in cell
cultures is treatment with antibiotics. In general, antibiotic
therapies do not result in long-lasting, successful elimination. Also,
the cytotoxic properties of antibiotics can cause undesirable side
effects on eukaryotic cells and can facilitate the development of
resistant mycoplasma strains. The Mynox® technique offers a number of
outstanding characteristics compared to other methods and products
used for mycoplasma elimination.
Mynox®
is the first biological reagent that actually eliminates mycoplasma by
killing them.
Mynox®
has been shown to be effective with only one treatment.
Mynox®
activity is based on its biophysical properties, making the
development of resistant strains highly unlikely.
Mynox®
is easily removed after the treatment.
Biological
Activity of Mynox® against Mycoplasmas
In
comparison to mammalian cells, mycoplasma lack a cell wall but are
encircled by a cytoplasmic membrane. Mynox® is a biological agent
that integrates into the mycoplasma membrane and compromises its
integrity. The result is an osmotic influx that leads to the complete
disintegration of the mycoplasma membrane. With mycoplasma eradication,
mammalian cells immediately return to their native morphology and
normal proliferation rates. To date, Mynox® has not been shown to
cause any changes in normal cell characteristics.
Application
Mynox®
is used for the elimination of Mycoplasma and Acholeplasma in cell and
virus cultures, and other biologicals. Mynox® is intended for
research only.
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General
Information
For
both biological and economical reasons, it is important to eliminate
mycoplasma from cell cultures being used for basic research, diagnosis,
and biotechnological
Importance
of Serum Concentration
The
mycoplasmacidal activity of Mynox® is affected by the concentration
of lipids and proteins in the reaction mixture, e.g. components in
fetal calf serum supplement. These ingredients competitively bind
Mynox® and prevent its binding to the mycoplasma membrane. Therefore,
the protocol for mycoplasma elimination in cell cultures was designed
for a specific standard cell culture medium, e.g. Dulbecco´s modified
Eagle´s medium (D-MEM) or RPMI1640. For the treatment of cell lines,
a protocol was designed that recommends a supplement of 5 % v/v fetal
calf serum. For virus stocks, it is
recommended that the
medium be almost free of supplemental serum during treatment.
Limits
of Mynox®
Mynox®
will not eliminate the cell penetrating Mycoplasma penetrans. Also,
due to the mitigating effect of serum, it is impossible to design a
specific protocol that is applicable for the treatment of biologicals
with high protein and lipid concentrations.
Effect
of Mynox®
on
Cell Proliferation
Similar
to all other products available for Mycoplasma inactivation, Mynox®
also shows a cytotoxic effect on adherent and nonadherent cell lines.
Our protocols were tested on numerous cell lines and found to have a
cytotoxicity between 10 and 80%, with enough viable cells recovered
for further subcultivation. Generally, higher proliferation rates as a
result of parasite removal will compensate for lost cell material.
Special
Precautions
Since
Mynox® works by biophysical means through association with the
mycoplasma membrane, the reagent needs direct contact with the
mycoplasma particle in order to be effective. Treatment of cell
clusters should be avoided. Mycoplasma are protected in intercellular
spaces as well as in pockets and clefts of the cell membrane, which
can prevent contact with the drug. We suggest using trypsin to detach
the cells from each other and to smoothen cell surfaces.
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Protocol
Guide
These
protocols have been designed with typical cell lines requiring
standard media. Minerva Biolabs does not guarantee that these
protocols will work in all laboratory situations. Modification of
these protocols maybe required per individual case.
Treatment
of Adherent Cell Lines
Preparation
of cells and the elimination mix
Transfer
1x104 to 1x105 freshly trypsinized cells into a
sterile cell culture flask or petri dish containing standard cell
culture medium with 200 µl Mynox®. The total volume of the culture
should equal 5 ml. The final FCS concentration is 5 % v/v. Ensure that
Mynox® is already present in the culture medium before adding cells.
Treatment
and Mynox® removal
After
2 hours of incubation under normal growth conditions, remove the
elimination mix by discarding the supernatant, then overlay the cells
with standard cell culture medium.
For
the most effective method, maintain the cells in the elimination mixture
for one entire passage (approximately 3 to 8 days) under normal growth
conditions. Then remove the medium containing
Mynox® and subculture the cells in standard medium as normal.
Treatment
of Suspension Cell Lines
Preparation
of cells and the reaction mix
Depending
on the proliferation rate, transfer 1x104 to 1x105 cells from a
suspension cell line to a centrifuge tube containing the elimination
mixture:
1.6
ml standard cell culture medium with 10 % v/v FCS
1.6
ml trypsin:EDTA (0.125%:5mM) in phosphate buffered saline (PBS)
200
µl Mynox®
Ensure
that the elimination mixture wets the complete inner surface of the
centrifuge tube.
Note:
Trypsin is needed to avoid cell aggregates. Replace the trypsin volume
with cell culture medium if cell separation can be achieved by other
techniques before adding them to the elimination mix.
Treatment
and
Mynox® removal
Shake
the mixture gently at room temperature for 30 min.
Pellet
the cells gently by centrifugation (600 x g, 5 min) and discard the
supernatant.
Resuspend
the cells in Mynox® free standard cell culture medium.
For
a more effective method, a subcultivation in the presence of Mynox® for
1 passage is possible: Resuspend
the cells in 5 ml cell culture medium containing 5% v/v FCS and 150 µl
Mynox®. Incubate the cells in this medium for 3 days in a culture flask
under normal growth conditions followed by separating the cells from the
elimination mixture by centrifugation, then subculture the cells in
Mynox® free growth medium.
Treatment
of Nonenveloped
Viruses
Preparation
of the cells and the reaction mix
Frozen
or fresh aliquots of cell and cell debris-free virus suspensions can be
treated. The virus titer does not influence the success of the treatment.
Mix
the following components in a sterile 1,5 ml reaction tube with
safety-lock tops:
125
µl virus stock, containing up to 8 % FCS
1
ml cell culture medium containing no FCS
100
µl Mynox®
Ensure
that the elimination mixture wets the complete inner surface of the
reaction tube.
Treatment
and Mynox® removal
Incubate
the elimination mixture at room temperature by gentle shaking for 2
hours.
The
reaction is stopped by diluting Mynox® 1:10 in culture medium. This
step can be beneficially done by using the elimination mixture to infect
a subconfluent, host cell culture for simultaneous propagation of the
mycoplasma free virus culture.
Final volume should be 10x that of the elimination mixture.
!!!
Test the host cell line for mycoplasma contamination prior to infection.
Treatment
of Enveloped Viruses
The
composition of the outer lipid membrane of enveloped viruses is comparable
to the mycoplasma membrane, the target of Mynox®. These
viruses are also vulnerable to Mynox® inactivation depending
on the treatment time and concentration used. To achieve mycoplasma-free
virus suspensions with an acceptable level for subcultivation, the initial
virus titer should be higher than 106 TCID50.
Preparation of cells and the reaction mix
Frozen
or fresh aliquots of cell and cell debris-free virus suspensions can be
treated.
Mix
the following components in a sterile 15 ml screw cap reaction tube :
0.5
ml virus stock, containing up to 8 % FCS
4.4
ml cell culture medium containing no FCS
100
µl Mynox®
Treatment
and Mynox® removal
Ensure
that the reaction mixture wets the complete inner surface of the
reaction tube. Incubate the reaction mixture at room temperature by
gentle shaking for 2h.
The
reaction is stopped by diluting Mynox® 1:10 in culture medium. This
step can be beneficially done by using the elimination mixture to infect
a subconfluent culture of the host cell line for simultaneous
propagation of the mycoplasma free virus culture.
Final volume should be 10x that of the elimination mixture.
!!!
Test the host cell line for mycoplasma contamination prior to infection.
There
mycoplasma elimination procedures can be repeated with the viruses
directly harvested from the host cell cultures to ensure that all
mycoplasma have been removed.
Treatment
of other
biologicals
For
samples with low protein and lipid concentrations, we recommend applying
Mynox® in a 1:50 dilution. But out of the diversity of possible
biologicals where Mynox® is applicable, we can only give general
recommendations for Mynox® applications. The most appropriate protocol
must be optimized for individual cases.
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Testing
for Mycroplasma
Mynox®
treated cell cultures and virus stocks should be subcultivated for two
additional passages without antibiotics and then assayed for
mycoplasma re-emergence to validate culture purity. For highly
sensitive detection of mycoplasma contamination, we recommend VenorGeM®
- Mycoplasma Detection Kit - that uses PCR*
technology
*
The Polymerase Chain Reaction (PCR) process is covered by patents
owned by Hoffmann-La Roche. Use of the PCR process requires a license.
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Appendix
Limited
Product Warranty
This
warranty limits our liability for replacement of this product. No
warranties of any kind, express or implied, including, without
limitation, implied warranties of merchantability or fitness for a
particular purpose, are provided. Minerva Biolabs shall have no
liability for any direct, indirect, consequential, or incidental
damages arising out of the use, the results of use, or the inability
to use this product.
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