Invivogen
Menu

Antimicrobial agents

General questions

Does InvivoGen have technical guidelines for the handling of cell culture contamination?

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

How do I avoid contamination of my cell culture?

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™.

How to identify the type of contaminant?

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.

What does InvivoGen offer to help protect my cells against different contaminants?

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. For more detailed information on how to deal with cell culture contamination, please download our practical guide dedicated to this topic: download our practical guide (.pdf).

Can antimicrobial agents be used during the initial cultures to prevent contamination before making my frozen stocks?

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.

Do you provide an anti-microbial agent that protects cells from all types of contaminants, meaning bacteria, fungi and mycoplasma?

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.

Do the anti-microbial agents you have in your catalogue interfere with selective antibiotics?

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

Bacterial contamination

How can I detect bacterial contamination?

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™.

What does InvivoGen offer to prevent bacterial contamination?

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.

What does InvivoGen have to offer to treat bacteria contaminated cell cultures?

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 have a bacterial contamination but cannot determine which bacterial strain has contaminated our cultures. What would be the best option to ensure treatment of my cells?

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.

Fungal contamination

How do I detect fungal contaminations in my cell culture?

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.

What does InvivoGen have to offer to prevent or eliminate fungal contamination?

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.

Can Fungin™ be used in combination with penicillin and streptomycin (Pen-Strep)?

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

Can Fungin™ be used in other media such as blood agar and MacConkey agar?

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

I have been treating my culture with Fungin™ for 7 days now but I am not sure that my culture is completely devoid of fungal contamination, what would you recommend I do?

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.

At what concentration would you recommend using Fungin™ to treat my cell culture?

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 contamination

How to determine if my culture is mycoplasma contaminated?

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™

What does InvivoGen offer to prevent mycoplasma contamination of cells?

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.

What does InvivoGen offer for the detection of mycoplasma contamination?

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.

Can you provide a brief description of InvivoGen’s mycoplasma detection kit - MycoStrip™ and how it detects mycoplasma?

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. For more detailed information, please check our cell culture contamination guide: Read our Pratical guide (.pdf)

Can you provide a brief description of InvivoGen’s mycoplasma detection kit - PlasmoTest™ and how it detects mycoplasma?

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.
For more detailed information, please check our cell culture contamination guide: Read our Pratical guide (.pdf)

What does InvivoGen have to offer to treat mycoplasma infected cells?

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.

In what case should we use Plasmocure™ instead of Plasmocin™ Treatment to treat mycoplasma-infected cells?

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).

What to do if we have mycoplasma contamination in Plasmocin™-resistant cells and Plasmocure™-resistant cells?

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 to use 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.

Is Plasmocin™ treatment effective on intracellular mycoplasma?

Plasmocin™ is active on both extracellular and intracellular mycoplasma.

I carry out the mycoplasma treatments in T25 flasks but do you know if it is better to carry them out in 6-well plates (treat a lower number of cells)?

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).

We obtained a negative result when doing PCR for the detection of mycoplasma in the supernatant of our cells after treating our cells with Plasmocin™. However, after a couple of weeks the culture was contaminated again.

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.

Is Plasmocin™ suitable for the elimination of mycoplasma in insect cells?

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

Sterile contamination - endotoxins

What are sterile contaminants and why would they be an issue for my cell cultures?

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. For more information on endotoxin contaminations, please check our cell culture contamination guide (.pdf).

What are the sources of endotoxin contaminations?

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.

Can you provide a brief description of HEK-Blue™ LPS Detection Kit 2 and how it detects LPS?

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).

How many tests can be performed with the HEK-Blue™ LPS Detection Kit 2?

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).

What is the sensitivity of the HEK-Blue™ LPS Detection Kit 2?

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

What is the acceptable FBS endotoxin level cut-off to use with HEK-Blue™ LPS Detection Kit 2?

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.

What is the stability on the opened HEK-Blue™ LPS Detection Kit 2?

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)

When using blood samples to detect the amount of LPS, how can we avoid non-specific activation of the NFkB response by NFkB-activating cytokines in the blood?

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).

Primocin™

Does Primocin™ contain Tetracycline?

No, Primocin™ does not contain Tetracycline.

Should Primocin™ be used for prevention or elimination?

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.

What is the preferred storage method for Primocin™?

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.

What is the difference between Primocin™ and Normocin™?

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™

Is Normocin™ necessary for cell culture?

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.

Can Normocin™ be used in hybridoma cultures?

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

What is the preferred storage method for Normocin™?

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.

What is the difference between Normocin™ and Plasmocin™ prophylactic?

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.

What is the difference between 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.

We need an antimicrobial agent for our cell culture to prevent all types of contamination however it cannot contain any Phenol Red (which would interfere with our assay). Does Normocin™ contain Phenol Red? If it does contain Phenol Red is there an alternative?

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™

What is the preferred storage method for Normocure™?

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.

Can Normocure™ be used to eliminate all types of bacteria (Gram+ and Gram– bacteria)?

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.

Plasmocin™ Treatment

When thawing Plasmocin™ treatment, one of the two 1ml aliquots was perfectly clear but the other was cloudy or turbid. When heating to 37°C the precipitation wouldn’t dissolve. Same goes for when sonicating the vial. The two aliquots have the same lot number. Any suggestions would be greatly appreciated.

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.

When treating cells with Plasmocin™ Treatment, should it be in media without gentamycin or fungizone™?

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.

What is the preferred storage method for Plasmocin™ Treatment?

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.

We have previously been treating our mycoplasma-contaminated cells with other anti-mycoplasma agents than those provided by InvivoGen, but it has been without success. We would therefore like to try your Plasmocin™ Treatment. Do you have any tips for the treatment of these cells?

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™

What is the preferred storage method for Plasmocure™?

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.

In what case should we use Plasmocure™ instead of Plasmocin™ Treatment to treat mycoplasma-infected cells?

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).

What to do if we have mycoplasma contamination in Plasmocin™-resistant cells and Plasmocure™-resistant cells?

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 to use PlasmocureTM 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.

Plasmocin™ Prophylactic

Do you have a CAS number for Plasmocin™ Prophylactic?

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

Are calcium salts present in Plasmocin™ prophylactic?

There are no calcium salts in Plasmocin™ prophylactic.

Does Plasmocin™ contain Tetracycline?

No, Plasmocin™ does not contain Tetracycline.

What is the preferred storage method for Plasmocin™ prophylactic?

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.

Fungin™

Does Fungin™ contain Amphotericin B?

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.

Can Fungin™ be used in combination with penicillin and streptomycin (Pen-Strep)?

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

Can Fungin™ be used in other media such as blood agar and MacConkey agar?

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

I have been treating my culture with Fungin™ for 7 days now but I am not sure that my culture is completely devoid of fungal contamination, what would you recommend I do?

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.

At what concentration would you recommend using Fungin™ to treat my cell culture?

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.

What is the preferred storage method for Fungin™?

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™

What is the principle of the MycoStrip™ assay?

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 immunochromatographic strip.

Is it necessary to include a positive and negative control for each sample tested when using MycoStrip™?

It is important to include positive and negative controls on a regular basis to monitor the reliability of your results and in case of troubleshooting.

Is the positive control provided in MycoStrip™ a source of mycoplasma contamination?

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

I have accidentally collected some cells with the supernatant that is to be tested, will this interfere with the assay?

There is no need to worry, the presence of mammalian cell DNA will not affect the detection assay.

At what stage can I freeze my sample for later analysis?

Samples can be stored as followed:
Cell culture supernatant: before starting the “preparation of samples” protocol, you can store the cell culture supernatant at -20°C until required.
Before use, completely thaw the sample at 37°C and mix well by vortexing to ensure all salts are dissolved.

Prepared sample: After performing steps 1 – 4 of the “preparation of samples” protocol, your sample can be stored at -20°C until required.

Processed sample: After performing steps 1 – 4 of the “detection assay” protocol, the processed samples can be stored at -20°C for up to 3 months.

I have a faint band for one of my samples and am unsure if this should be considered as positive or not for mycoplasma contamination. What would you recommend?

If you observe a faint band for one of your samples, we consider this to be a positive result for mycoplasma contamination. We recommend to confirm this result using one of the following tips:
• Concentrate your sample: begin with a larger volume of supernatant and/or repeat steps 2-3 in the “preparation of samples” protocol. Re test the concentrated sample using MycoStrip™.
• Continue to grow the culture for an additional 48 hours before re-testing using MycoStrip™.
• Use sterile water to resuspend the sample instead of PBS (to increase the sensitivity of the test).

What is the sensitivity of the MycoStrip™ test?

MycoStrip™ is highly sensitive having been validated to detect as low as 102 genome copies per ml.

Does MycoStrip™ detect certain species of bacteria as well as mycoplasma in cell cultures?

MycoStrip™ does not detect any of the phylogenetically related microorganisms, such as Clostridium, Lactobacillus, and Streptococcus. Likewise, the waterborne germ Burgholderia is not detected. The specificity of MycoStrip™ has been assessed on DNA extracts of Mollicutes, non-Mollicute bacteria, and eukaryotic cell/tissue samples. Additionally, the cross-reactivity with eukaryotic DNA was not observed. For a complete list of all species detected and tested, please visit the following webpage

What type of equipment is required to perform the MycoStrip™ assay?

Contrary to other mycoplasma detection kits, only a heat block is needed!

How long does it take to perform the assay?

The sample preparation requires only 10 minutes and the detection assay takes 60 minutes including 10 minutes hands on time. Results are visualized as a band within 5 minutes.

What sample size is required to perform the assay?

The MycoStrip™ 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).

What are the advantages of the MycoStrip™ detection system?

MycoStrip™ is a robust, sensitive, and extremely simple method for the detection of mycoplasma. The speed and convenience of MycoStrip™ 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.

What are the storage conditions required for the MycoStrip™ kit?

The kit is shipped at room temperature.
Upon receipt, store all components at -20°C.
All components, including the strips, remain stable for 12 months when properly stored.

Our cells to be tested with the MyocStrip™ kit were cultured with antibiotics. Could the use of antibiotics interfere with the results? Is it better to use antibiotic-free media when performing the assay?

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

Is there an advantage to lyse the sample?

There is no increase to sensitivity however this step will kill the mycoplasma avoiding the handling of live mycoplasma.

Is it possible to use water instead of PBS to make the prepared sample?

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

I do not have a heat block in the lab, what would you recommend to use instead?

If you do not have a heat block, you can use a thermocycler or water bath instead. Please note that the use of a water bath can introduce new sources of contamination, therefore we recommend to use parafilm to protect your sample.

I have left my prepared sample for over an hour on the heat block and wonder if I can still use this sample?

Exceeding the 40-minute incubation at 65°C significantly impacts the results of the assay and therefore, we do not recommend to use this sample.

PlasmoTest™

What are the advantages of using InvivoGen’s mycoplasma detection kit - PlasmoTest™?

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.

Does InvivoGen’s mycoplasma detection kit - PlasmoTest™ detect only live mycoplasma?

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

Does PlasmoTest™ detect the presence of bacteria in cell cultures?

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.

Is there an optimal wavelength to read the plates?

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.

Do you have some recommendations regarding the amount of cells used for the testing?

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.

Our sample that needs testing should not be heated however you state in the protocol for the PlasmoTest™ that all samples must be heated, can you tell me why that is?

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.

I couldn’t find any information in the technical data sheet regarding contents and concentration of the positive and negative control provided in the PlasmoTest™ kit. Can you provide any information on this?

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

We got some false positive results and I wonder if there are some supplements such as DMSO, FBS, Trypsin or PBS which could affect the results? Do you have some experience with this?

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.

When performing assays with PlasmoTest™, all wells are blue, even the negative control, do you have an explanation for this?

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

What are the expected ODs for the positive and negative controls?

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

We accidentally left the supernatant to be tested 2 days at room temperature. Do you know if this could falsify the test results?

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.

What split ratio would you recommend for the HEK Blue™-2 cells once the vial has grown confluent?

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.

Can you confirm that I should see powder in both the positive and the negative control?

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.

Can the PlasmoTest™ kit be used for plasma or serum samples? If so, what provides the best results, plasma or serum samples?

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.

HEK-Blue™ LPS Detection Kit 2

How many tests can be performed with the HEK-Blue™ LPS Detection Kit 2?

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).

What is the sensitivity of the HEK-Blue™ LPS Detection Kit 2?

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

What is the acceptable FBS endotoxin level cut-off to use with HEK-Blue™ LPS Detection Kit 2?

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.

What is the stability on the opened HEK-Blue™ LPS Detection Kit 2?

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)

When using blood samples to detect the amount of LPS, how can we avoid non-specific activation of the NFkB response by NFkB-activating cytokines in the blood?

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)

Customer Service
& Technical Support
Contact us
Shopping cart is empty