Frequently asked questions

Yes, InvivoGen provides a free guide dedicated to this topic: Read our Pratical guide (.pdf).

Firstly, ensure that you are working in a sterile environment and using proper aseptic techniques. It is also important to avoid talking over your cells and sneezing. Secondly, quarantine any incoming cell cultures until these have been confirmed free of contamination. Thirdly, monitor your cell cultures for contamination on a regular basis by optical microscopy and detection kits. For example, InvivoGen provides MycoStrip™ and PlasmoTest™ for the detection of mycoplasma contamination in cell cultures. Lastly, you can use antibiotic cocktails, such as those offered by InvivoGen, specifically designed for taking a preventive strike against microbes that would be difficult to detect in new cultures (i.e. primary cells, or cloning), such as Plasmocin® prophylactic, Normocin™, Primocin® or Fungin™.

Bacteria and fungi can usually be identified by optical microscopy. Their fast growth rate allows their detection by the naked eye as early as 48 hours (i.e. over the weekend), the contaminated cultures appearing turbid or spotty. Subsequently, identification of these microorganisms can be performed with testing kits. Mycoplasma in cell cultures cannot be detected visually, not even by optical microscopy. Hence, these microbes can go unnoticed for long periods and can only be identified using dedicated assays. Detection techniques (such as MycoStrip® and PlasmoTest™) can be used in tandem to ensure accurate analysis, especially to rule out false positives or clarify ambiguous results.

All depends on the cell line you have in culture and if you are looking to prevent infection from a specific type of contaminant or a broader range of contaminants.
For example, if you are specifically looking to prevent fungal contaminations from your cell cultures, we would recommend using our Fungin™. If you are looking to prevent mycoplasma contamination, we have Plasmocin® prophylactic.
If on the other hand you are looking for to prevent infection from a broad range of contaminants, we would recommend Normocin™ which is designed to prevent cell lines from mycoplasma, bacterial and fungal contaminations. We also have Primocin® which is specifically designed to offer complete protection of primary cells from microbial contamination.

Yes, it is even recommended to use either Plasmocin® prophylactic or Normocin™. 
Please note however that it is necessary to check that the cells are not contaminated beforehand.

We actually provide two antibiotics for this purpose, Normocin™ and Primocin®. Both antibiotics provide complete protection from microbial contamination, however, Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.

No, they do not interfere with common selective antibiotics such as G418, Blasticidin, Puromycin, Hygromycin B, and Zeocin®.

In the early stages of contamination, bacteria can be mistaken for cellular debris as they are much smaller (1-10 μm) than eukaryotic cells (10-100 μm). Therefore, it is important to check your cell cultures under a light microscope using phase contrast (100x - 400x). If you observe the abnormal presence of small black dots, rods, spirals, either alone, in chains, or clusters, your culture is probably contaminated. Because of their fast growth rate, bacteria cause a change in the culture medium in just 48 hours, making the contamination clearly visible with the naked eye from 10^5 cfu/ml. The culture medium appears cloudy, and if it contains phenol red, a rapid colour change from red to yellow indicates a decrease in pH, a consequence of bacteria metabolism. The culture environment is no longer suitable for eukaryotic cells leading eventually to their death. These cultures should be discarded, or if irreplaceable, treated with InvivoGen’s antibiotic cocktails such as Plasmocin® Treatment, Normocure™ or Plasmocure™.

InvivoGen provides a range of anti-bacterial agents to prevent contamination of your precious cell lines. First we have Normocin™ which is an innovative formulation of three antibiotics active against mycoplasma, bacteria (Gram+ and Gram-), and fungi, including yeasts. It is widely used and cited as a “routine addition” to cell culture media to prevent bacterial contamination.
Next we have Primocin® which contains four compounds, with three of these blocking DNA and protein synthesis in Gram+ bacteria, Gram- bacteria, and mycoplasma. Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.
Finally, we also have Plasmocin® prophylactic which contains two potent bactericidal components: one that acts on protein synthesis machinery, and one that acts on DNA replication. Although originally designed to target mycoplasmas, it is also active against other types of bacteria.

Normocure™ is the best weapon to save your valuable cell lines from Gram+ and Gram- bacteria, especially non-fermenting Gram- bacteria that are resistant to Pen-Strep and Normocin™. Normocure™ is a cocktail of three components belonging to different antibiotic families. After the first passage, > 99% of bacterial contaminants are eliminated. The targets of these antibiotics are absent in eukaryotic cells, ensuring Normocure™’s low cytotoxicity.
Please note that we also have Plasmocin® Treatment and Plasmocure™ which can be used for bacterial contaminations. Although these products are designed to target mycoplasmas, they are also active against certain types of bacteria.

We would highly recommend using Normocure™ which is a broad-spectrum antibacterial agent highly effective against Gram+ and Gram- bacteria. Cell cultures contaminated with bacteria from the environment, such as Staphylococcus species and Achromobacter species, can be efficiently cured by Normocure™ treatment.

Hyphae can be detected by the naked eye or a light microscope depending on their size and growth stage. Yeasts are the smallest form (3-10 μm) of fungi. They can be seen using a light microscope.
In the case of substantial contamination, colonies form as molds floating on the surface. In this case, do not open the vessel and discard the cultures to avoid spreading spores. Sometimes, the medium pH may increase, resulting in phenol-red containing media to appear pink.

For both the protection and elimination of fungal contaminations in your cell cultures, InvivoGen provides Fungin™ which is a highly referenced antimycotic reagent. Fungin™ kills the different forms of fungi (yeasts, hyphae, and molds) by disrupting ionic exchange through the cell membrane.

Fungin™ may be added to media containing commonly used antibacterial agents, such as Pen-Strep.

You should have no problem using our antifungal reagent, Fungin™ in other media such as blood agar and MacConkey agar.

We would recommend continuing treatment on one half of the culture for at least another 3 – 4 days. On the other half of the culture, change the medium with fresh medium that no longer contains any antibiotics (no Fungin™ or Pen-Strep) and spread 1 mL of the culture medium on Sabouraud agar to determine if the fungal contamination is still present. For this you should leave the petri dish at 20 - 25°C and check after 3 – 5 days if fungi appears.

On the technical data sheet we state that Fungin™ should be used at 50 µg/ml but we would recommend using it in parallel at 75µg/ml (seeing as it is not very toxic for cells) to estimate the best concentration for fungi removal. The treatment should last 5 – 10 days.

Mycoplasma in cell cultures cannot be detected visually, not even by optical microscopy. Hence, these microbes can go unnoticed for long periods and can only be identified using dedicated assays, ie MycoStrip™ and PlasmoTest™

Plasmocin® prophylactic was specially designed for the prevention of mycoplasma contaminations. It contains two potent bactericidal components: one that acts on protein synthesis machinery, and one that acts on DNA replication.
We also provide Normocin™ and Primocin® which provide complete protection from microbial contamination (such as mycoplasma, bacteria and fungi).
Please note however that Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.

InvivoGen offers two different kits for the detection of mycoplasma, PlasmoTest™ and MycoStrip™. PlasmoTest™ and MycoStrip™ allow rapid and accurate detection of cell culture contamination by mycoplasma as ‘routine’ and ‘occasional’ procedures, respectively.
PlasmoTest™ is a cell-based colorimetric assay which, in the presence of mycoplasma contaminated samples, induces the secretion of Alkaline Phosphatase which is easily detected in culture medium. The hands-on time is about 30 minutes and the results are obtained after an overnight incubation. This kit is specially designed for labs doing a lot of cell culture and can be established as a routine procedure in the lab. The advantage of this kit is that it is highly cost-effective as the critical component of the PlasmoTest™ system is an engineered cell line that can be propagated to make frozen stocks for unlimited use. Once you have the PlasmoTest kit you only need to purchase the HEK-Blue Detection media, reducing drastically the cost per sample.
Additionally, InvivoGen has designed a new and innovating mycoplasma detection assay, MycoStrip™, based on isothermal PCR. Results are visualized as a band on an lateral flow detection strip within 5 minutes. This kit is specially designed to provide the user with minimal hands on time and rapid and clear results – with no need for calculation, distinguishing similar colors, or running of an agarose gel. Additionally, the only equipment needed to perform the assay is a heat block.

Detection of mycoplasma by InvivoGen’s MycoStrip™ is based on isothermal PCR, a robust technique that exponentially amplifies DNA at a constant temperature of 65°C. Using our proprietary Reaction Mix, the 16S rRNA gene in the most commonly found mycoplasma species in cell culture, accounting for 95% of contamination, is targeted and amplified. Results are rapidly visualized as a band on an lateral flow detection strip.

PlasmoTest™ is a cell-based colorimetric assay that exploits the ability of Toll-like receptor 2 to recognize mycoplasmas and to induce a signalling cascade leading to the activation of NF-κB and other transcription factors. In the presence of mycoplasmas, TLR2 expressed on the surface of HEK-Blue™-2 cells activates these transcription factors which in turn induce the secretion of sAP (secreted alkaline phosphatase), a reporter protein easily detectable by the purple/blue coloration of the HEK-Blue™ Detection medium.

Mycoplasma contamination can be efficiently and rapidly eliminated with the Mycoplasma removal agents, Plasmocin® or Plasmocure™. They combine two antibiotics that act through different mechanisms and allow mycoplasma eradication in only 2 weeks.

Plasmocin® and Plasmocure™ are cocktails of different antibiotics, both of which will allow you to get rid of your mycoplasmas completely. Please note however that we recommend starting treatment with Plasmocin®.
(We recommend using Plasmocure™ only in case of resistance to Plasmocin®, which is extremely rare).

If both Plasmocin® and Plasmocure™ did not work then it seems like your sample is too contaminated.
The best option, in this case, is to dilute the cells as much as possible at the next passage (this way you will also dilute the quantity of contaminants).
Then we recommend using Plasmocure™ at different concentrations (to be sure that it is not too toxic for the cells) for 2 weeks. After the 2 week treatment, split the cells in two and on one half continue the treatment for another week and on the other half, wait three days without using any antibiotics and then check for the presence of the contaminant, using for example PlasmoTest™. If you are still contaminated, please contact us.

Plasmocin® is active on both extracellular and intracellular mycoplasma.

Treatment in a T25 flask is suitable.
Please note that the treatment is most effective when the cells are “passaged” as much as possible (to remove mycoplasma and add fresh Plasmocin® or Plasmocure™) and also when the cells are not too confluent (60 - 70% max).

It seems like all mycoplasmas were not killed following the treatment. To prevent this from happening again, we recommend dividing your culture in half after the two weeks of treatment with Plasmocin®.
On one half you continue the treatment for another week (without interruption) to prevent the mycoplasmas from developing again if they are still present in a basal manner. On the other half, we recommend to stop the treatment and grow your cells 3 days without Plasmocin® or any antibiotics at all.
After the 3 days of culture, you can then perform your mycoplasma detection test which should detect any residual mycoplasma that was left after the two week treatment.

Plasmocin® is suitable for Sf9 cells, and should suit other insect cells (but unfortunately we do not have additional data on other insect cells).

Endotoxins, also known as lipopolysaccharides (LPS) or lipoglycans, are a major cell wall component of Gram-negative bacteria. Endotoxins are potent inducers of inflammatory responses both in vitro and in vivo. Endotoxin contaminations are a major concern for cell cultures and production of injectable drugs. Thus, extra-care needs to be taken with solutions and reagents that are sterile but may still contain bacterial components, and monitoring for the presence of endotoxins in cell culture reagents is crucial.

Sources of endotoxins include media, sera, water, buffers and other cell culture reagents, such as trypsin. To avoid having endotoxin contaminants, we recommend to ask for manufacturer certification of endotoxin levels, or test before use using our HEK-Blue™ LPS Detection kit 2.

The HEK-Blue™ LPS Detection Kit is a cell-based colorimetric assay for the detection of biologically active endotoxin. This kit is based on the ability of TLR4 to recognize structurally different LPS from gram-negative bacteria.
The presence of minute quantities of LPS, starting as low as 0.01 EU/ml, are detected by the HEK-Blue™-4 cells leading to the activation of NF-κB. Using HEK-Blue™ Detection, a specific detection medium, NF-κB activation can be observed with the naked eye or quantified by reading the OD at 650 nm.
You can find further details on the following document: HEK-Blue™ LPS Detection Kit (.pdf).

With the LPS detection kit 2, we provide 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent).
This should be sufficient to perform assays in approximately five 96-well plates (480 wells).

The LPS detection kit is highly sensitive and can detect as little as 0.01 EU/ml.

The use of some FBS might affect the functionality of HEK-Blue™-4 Cells as they may contain endotoxins which would interfere with your assay. Make sure the FBS used is endotoxin-free. To avoid having endotoxin contaminants, we recommend to ask for manufacturer certification of endotoxin levels before purchasing the FBS to ensure it is endotoxin-free.

The cell line should be propagated upon receipt to make your frozen stocks for further use (no expiry date as the cells can be kept a very long time in liquid nitrogen). Regarding the rest of the kit, it should remain stable for 1 year (providing that it is stored in the correct conditions)

There are 2 ways to avoid this nonspecific activation:
- You can either use our parental cell line as a negative control, the HEK-Blue™ Null1 cells (this cell line only expresses the NFkB inducible system). Therefore, any non-specific activation of NFkB will also be detected with the HEK-Blue™ Null1 cells.
- Otherwise, you can antagonize the TLR4 response with the Ultrapure LPS-RS which is an antagonist of LPS. If with this antagonist you eliminate the signal, this will then confirm that it is a specific TLR4 activation of your sample.
The reference of this antagonist is the following: LPS-RS ultrapure (#tlrl-prslps).

No, Primocin® does not contain Tetracycline.

Primocin® is a preventive antibiotic, active against both Gram+ and Gram- bacteria, mycoplasma and fungi. In other words, Primocin® will NOT induce the elimination of microbial contamination in already infected cells.

Primocin® is shipped at room temperature.
Upon receipt it can be stored at 4°C for 3 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

Both antibiotics provide complete protection from microbial contamination however Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.

Yes, Normocin® is one of the antibiotics we use as a standard antimicrobial agent.
Our cell lines have been developed with this antimicrobial agent so we prefer to use it in house.

Yes, in house we use Normocin® for hybridoma culture.

Normocin® is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi. Plasmocin® prophylactic on the other hand is a cocktail of 2 different antibiotics, it is a specific anti-mycoplasma reagent.

Both antibiotics provide complete protection from microbial contamination however Primocin® was specifically designed for the protection of primary cells whereas Normocin® is recommended for cell lines.

Normocin® does contain Phenol Red. If you cannot use an additive with Phenol Red and would like a broad-spectrum antibiotic/antimycotic/anti-mycoplasma agent, you can use our Primocin®.

Normocure™ is shipped at room temperature.
Upon receipt it can be stored at 4°C for 3 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

Yes, Normocure™ is a broad-spectrum antibacterial agent highly effective against Gram+ and Gram- bacteria. Cell cultures contaminated with bacteria from the environment, such as Staphylococcus species and Achromobacter species, can be efficiently cured by Normocure™ treatment.

The cloudy precipitate is one of the two antibiotics in Plasmocin® coming out of solution after freeze-thawing. 
The antibiotic is basically at the limit of its solubility at the concentration provided and thus freeze-thawing causes it to come out of solution. 
We don’t recommend sonicating. Letting it sit at 37 °C for 30 minutes - 1 hour and then vortexing for a minute or two should get it back into solution. 
Plasmocin® is stable for 1 week at 37 °C so there is no concern about heating it for an hour to get the precipitate back in solution. Alternatively you can make a more dilute stock (12.5 mg/ml) by making a 1:1 dilution with sterile water thus increasing the volume in which the antibiotic is dissolved in.

We do not recommend using Plasmocin® with other antibiotics (such as gentamycin and fungizone™) as this may interfere. Plasmocin® can be toxic in some cells therefore we prefer not using additional antibiotics with this treatment that could end up overwhelming sensitive cell lines.

Plasmocin® Treatment is shipped at room temperature.
Upon receipt it can be stored at 4°C for 1 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

If possible, we highly recommend starting treatment on a fresh batch of cells that haven’t been treated with another anti-mycoplasma agent to be sure that your cells aren’t resistant to one of the antibiotics present in Plasmocin® Treatment.

Plasmocure™ is shipped at room temperature.
Upon receipt it can be stored at 4°C for 12 months or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions and at a concentration of 100 mg/ml.

Plasmocin® and Plasmocure™ are cocktails of different antibiotics, both of which will allow you to get rid of your mycoplasmas completely. Please note however that we recommend starting treatment with Plasmocin®. (We recommend using Plasmocure™ only in case of resistance to Plasmocin®, which is extremely rare).

If both Plasmocin® and Plasmocure™ did not work then it seems like your sample is too contaminated.
The best option, in this case, is to dilute the cells as much as possible at the next passage (this way you will also dilute the quantity of contaminants).
Then we recommend using Plasmocure™ at different concentrations (to be sure that it is not too toxic for the cells) for 2 weeks. After the 2 week treatment, split the cells in two and on one half continue the treatment for another week and on the other half, wait three days without using any antibiotics and then check for the presence of the contaminant, using for example MyocStrip™ or PlasmoTest™.
If you are still contaminated, please contact us.

There is no CAS number for Plasmocin® Prophylactic seeing as it is a cocktail of different antibiotics

There are no calcium salts in Plasmocin® prophylactic.

No, Plasmocin® does not contain Tetracycline.

Plasmocin® prophylactic is shipped at room temperature.
Upon receipt it can be stored at 4°C for 1 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

No, Fungin™ does not contain Amphotericin B. Unlike Amphotericin B, Fungin™ is a highly stable compound and it does not need to be dissolved in toxic deoxycholate.

Fungin™ may be added to media containing commonly used antibacterial agents, such as Pen-Strep.

You should have no problem using our antifungal reagent, Fungin™ in other media such as blood agar and MacConkey agar.

We would recommend continuing treatment on one half of the culture for at least another 3 – 4 days. 
On the other half of the culture, change the medium with fresh medium that no longer contains any antibiotics (no Fungin™ or Pen-Strep) and spread 1 mL of the culture medium on Sabouraud agar to determine if the fungal contamination is still present. For this you should leave the petri dish at 20 - 25°C and check after 3 – 5 days if fungi appears.

On the technical data sheet we state that Fungin™ should be used at 50 µg/ml but we would recommend using it in parallel at 75µg/ml (seeing as it is not very toxic for cells) to estimate the best concentration for fungi removal. The treatment should last 5 – 10 days.

Fungin™ is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions and at a concentration of 10 mg/ml.

MycoStrip® 2.0 detects mycoplasma using loop-mediated isothermal amplification (LAMP) PCR, a robust amplification method performed at a constant temperature of 65°C. The assay targets a conserved region of the 16S rRNA gene within mollicute genera using a proprietary reaction mix. Amplified DNA is then visualized as a band on an immunochromatographic strip, enabling rapid and easy result interpretation.

MycoStrip® 2.0 has been upgraded with an optimized, high-performance enzyme that enhances the overall efficiency of the amplification reaction. This improvement directly translates into better assay performance compared to the previous version of MycoStrip®. 

As a result, MycoStrip® 2.0 achieves detection of 10 - 100 genome copies (GC)/µl (~CFU/ml) in just 30 minutes, compared to 40 minutes required with the old version. Under optimal conditions and by extending the incubation time to 50 minutes, MycoStrip® 2.0 can detect as little as 1 GC/µl.

The updated reaction mix also provides a broader temperature tolerance, making the assay less sensitive to temperature fluctuations and therefore more robust across different laboratory conditions. As a result, the assay can be performed using a variety of standard heating devices, such as a water bath, dry bath, or thermocycler.

In addition, MycoStrip® 2.0 offers expanded detection coverage, including additional mollicute species, such as Ureaplasma and Spiroplasma genera, allowing detection of a wider range of potential contaminants. 
 

The assay is highly sensitive and can detect little as 1 GC/µl when incubated for 50 minutes. Longer incubation will not improve detection.

Sensitivity is now expressed in genome copies per µl (GC/µl) in alignment with updated edition 12.2 of the European Pharmacopoeia, which favors molecular quantification methods over culture-based measurements.

While CFU/ml (colony-forming units) reflects the number of viable organisms capable of forming colonies, GC/µl measures the amount of mycoplasma DNA present in a sample. This molecular approach provides a more direct and sensitive assessment of contamination, including both live and dead organisms.

To relate these two units, InvivoGen performed rigorous in-house studies using digital PCR (dPCR) on the most common mycoplasma contamination species. These results, together with external data, show that the ratio between GC and CFU varies depending on the species and method used, but is generally in the same order of magnitude.

Based on these internal validations, InvivoGen considers that 1 CFU/ml is approximately equivalent to 1 GC/µl for the most common mycoplasma contaminants, providing a practical reference for comparing molecular and culture-based measurements.

No. MycoStrip® 2.0 is highly specific for mycoplasma and does not detect phylogenetically related bacteria, such as Clostridium, Salmonella, or Streptococcus, nor waterborne organisms like Achromobacter. The assay specificity has been validated using DNA from Mollicutes, non-Mollicute bacteria, and eukaryotic cells/tissues, with no cross-reactivity observed with non-target DNA.

No. The presence of mammalian cells or their DNA does not interfere with mycoplasma detection.

Only a heating device is needed (e.g., thermocycler, heat block, dry bath, or water bath).

You can use a heat block, dry bath, or water bath. However, water baths may increase contamination risk.

• Hands-on time: 15 minutes
• Total time: 45 minutes

During the sample preparation step, heating the supernatant at 95°C for 5 minutes is optional. Although it does not increase sensitivity, it is recommended to inactivate mycoplasma and minimize contamination risks.

While not required for every single sample, it is strongly recommended to include positive and negative controls on a regular basis. This helps ensure the reliability of results and facilitates troubleshooting if needed.

The kit is shipped at room temperature.
Upon receipt, store all components at -20°C.
All components, including the strips, remain stable for 18 months when properly stored.

Samples can be stored at different stages of the workflow:

• Cell culture supernatant (before sample preparation):
  Can be stored at -20°C. Before use, thaw completely at 37°C and vortex to ensure proper mixing and salt dissolution.
• Prepared sample (after steps 1–4 of sample preparation):
  Can be stored at -20°C until needed.
• Processed sample (after steps 1–4 of the detection assay):
  Can be stored at -20°C for up to 3 months.

• Extend the 65°C incubation to 50 minutes for increased sensitivity. If a faint band remains faint after 50 minutes, it is considered negative.
• Alternatively, grow cells for 48 hours to achieve 100% confluency and re-test.
• Do not vortex the mix before the 65°C incubation step, as this can inhibit the reaction.

• Avoid repeated freeze–thaw cycles. The positive control as well as the Reaction Mix should be aliquoted upon first use and stored appropriately to minimize degradation.

• Do not vortex the reaction mix vigorously (it contains the enzyme).

• Do not leave the assembled reaction at room temperature for too long. Since the system is not warm-start, the reaction can still begin at room temperature. Keep this time below 15 minutes and transfer the reaction to 65°C immediately.

• Ensure the heating device is set to 65°C (±5°C).

No, the use of antibiotics on your cells will not interfere with the assay.

The positive control is not a source of mycoplasma contamination; it only contains a fragment of DNA.

The MycoStrip® 2.0 test requires 1 mL of cell culture supernatant from suspension or adherent cell cultures prior to passaging, or cell culture media constituents such as fetal bovine serum (FBS).

MycoStrip® 2.0 is a robust, sensitive, and extremely simple method for the detection of mycoplasma. The speed and convenience of MycoStrip® 2.0 allows for routine testing of cells in culture and commonly used constituents of complete media (e.g. FBS). It requires minimal hands-on-time with rapid results which are easily interpreted – no need for calculation, distinguishing similar colours, or running of an agarose gel.

Yes, it is possible to use nuclease free water instead of PBS.

No. Exceeding the recommended incubation time (maximum 60 minutes at 65°C) can significantly affect assay performance. We therefore do not recommend using samples that have been incubated for longer than specified.

No. Reagents from MycoStrip® and MycoStrip® 2.0 are not interchangeable.

Yes. MycoStrip® 2.0 detects both live and dead mycoplasma. This provides a more comprehensive assessment of contamination, as methods that detect only viable organisms may underestimate its presence. Detecting dead mycoplasma can still indicate a recent contamination event or the persistence of residual DNA. This is particularly important for quality control, where all traces of contamination must be taken into account.

This typically indicates contamination. To prevent this, use filtered tips and handle the positive control last.

The most common mycoplasma detection test used is PCR, but this technique has its drawbacks, notably if you do not perform PCR regularly in your lab this might be an issue due to the stability of the taq polymerase, sterility of all other PCR reagents such as dNTPs, buffer...
PlasmoTest™ is the first cellular assay for the visual, colorimetric detection of mycoplasma contamination in cell cultures. It requires only basic cell culture knowledge and a hands-on time of under 1 hour with results after overnight incubation. Most importantly, a positive result indicates the presence of a cell culture contaminant so there are no false positives.

PlasmoTest™ detects the presence of mycoplasma lipoproteins, thus it indicates the presence of both live and dead mycoplasma in the cell culture.

PlasmoTest™ relies on the activation of TLR2. Therefore, it can detect both mycoplasma and bacteria contaminants. However, while the mycoplasma cannot be detected by the naked eye, bacteria contamination is visible and leads to a decreased pH (change of medium colour to yellow) and medium turbidity due to bacterial growth.

Any wavelength between 620 nm and 655 nm is optimal to read the absorbance. Please note that for readers that cannot get to this part of the spectrum, you can get a signal from 600 – 680 nm. Based on our analysis of QUANTI-Blue with and without SEAP, looking at absorbance from 350 – 700 nm we find that 600 nm up to 680 nm will yield a signal, however there will be a lower saturation and potentially a higher background

It is important to collect a few cells with the supernatant as this will enable the detection of intracellular mycoplasma, if there are any as well as the extracellular mycoplasma which is in the supernatant.

All samples must be heated in order to get rid of any potential Alkaline Phosphatase present in the serum of your supernatants to be tested which can interfere with your results. The heating step also allows the elimination any remaining mycoplasma in this sample to avoid spreading of the contamination. You can however try testing just the test medium used for cell culture (without cells) and resuspended HEK-Blue™ detection. If the medium does not turn blue then you can skip the heating step but please be very careful with this potentially contaminated sample.

We cannot disclose any information on these controls but please be assured that the positive control is not live mycoplasma.

When developing PlasmoTest™ we have shown that some Trypsin or serum are able to induce a TLR2 response (from sterile contaminations) but never from PBS. DMSO over 1/1000 will be toxic for the HEK Blue cells which can induce relatively high background noise. It is useful to test the medium alone in parallel with your supernatant as a negative control.

There are 3 possible explanations as to why you observe a blue colour in all wells: 
1. It could be due to the presence of Alkaline Phosphatase (AP) in the culture medium. To see if this is the case, there is a very simple test that can be performed. Just add 50 µL of the medium used for cell culture (without cells) and 200µL of the resuspended HEK-BlueTM detection. If the medium turns blue then it is due to the AP in the serum of the media. In this case, you must heat up the culture medium to eliminate the AP and redo the same test. At this point the test must not be blue (medium + detection medium should give a negative result).

2. If the medium does not react with the HEK-BlueTM detection medium, then the problem might be due to the cells (HEK-BlueTM -2 cells) which are contaminated. In this case, the best option would be to start from fresh with a new vial of cells (if you have remaining stocks). Also, please note that it is best to use a pipet with filter-tips to avoid any contamination of the cell suspension.

3. It could be due to improper handling of cells before the test. Here are a few tips in order to lower the background level (to limit the activation of NFkB before stimulation) and therefore limit the risks of false positive results:
- use pre-warmed PBS to wash cells
- use heat inactivated FBS (some lots of FBS, although sterile, contain microbial debris that may activate the HEK-Blue™-2 cells)
- do not centrifuge cells prior to stimulation
- do not use trypsin

• DO (positive control): 0.9 – 1.2
• DO (negative control): < 0.2

If the sample was sterile and kept sterile at room temperature your results will be the same.
However it is recommended to freeze your samples at -20°C (for several days) if they can’t be tested right away.

The split ratio will depend on when you expect confluency. Typically, the doubling time of HEK-Blue™ cells is approximately 24 hours.
Therefore, if you use a split ratio of 1:2 (50%) into a new flask, cells should be confluent the following day. If you use a split ratio of 1:4 (25%) you can expect the cells to be confluent after 2 days.

Actually, the positive control is a translucid lipidic film that is often difficult to observe with the naked eye. The negative control on the other hand is a visible powder.

We have little experience testing plasma and serum samples however it has been performed on our HEK-Blue™-2 cells in the PlasmoTest™ kit. The results show that when compared to using standard samples (in DMEM), serum samples give a single log difference (example: in serum we detect up to 10^4 UFC/ml of Mycoplasmae Fermentans whereas in DMEM we detect up to 10^3 UFC/ml of M. Fermentans).
On the other hand, we found a 3-log difference between DMEM and plasma samples (example: in plasma we detect up to 10^6 UFC/ml of M. hominis whereas in DMEM we detect up to 10^3 UFC/ml of M. hominis). This is why we would recommend using serum samples over plasma samples.

With the LPS detection kit 2, we provide 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent). This should be sufficient to perform assays in approximately five 96-well plates (480 wells).

The LPS detection kit is highly sensitive and can detect as little as 0.01 EU/ml.

The use of some FBS might affect the functionality of HEK-Blue™-4 Cells as they may contain endotoxins which would interfere with your assay. Make sure the FBS used is endotoxin-free.
To avoid having endotoxin contaminants, we recommend to ask for manufacturer certification of endotoxin levels before purchasing the FBS to ensure it is endotoxin-free.

The cell line should be propagated upon receipt to make your frozen stocks for further use (no expiry date as the cells can be kept a very long time in liquid nitrogen).
Regarding the rest of the kit, it should remain stable for 1 year (providing that it is stored in the correct conditions)

There are 2 ways to avoid this nonspecific activation:
- You can either use our parental cell line as a negative control, the HEK-Blue™ Null1 cells (this cell line only expresses the NFkB inducible system). Therefore, any non-specific activation of NFkB will also be detected with the HEK-Blue™ Null1 cells.
- Otherwise, you can antagonize the TLR4 response with the Ultrapure LPS-RS which is an antagonist of LPS. If with this antagonist you eliminate the signal, this will then confirm that it is a specific TLR4 activation of your sample.
The reference of this antagonist is the following: LPS-RS ultrapure (#tlrl-prslps)