Antimicrobial agents

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. 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).

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

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)

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

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.

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.

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.

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

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

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.

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).

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

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

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

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.

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).

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.

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.

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

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

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

If you do not have a thermocycler, you can use a calibrated heatblock or a water bath instead. Please note that the use of a water bath can introduce new sources of contamination.

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.

As isothermal PCR is a very sensitive method, heatblock must be precisely at 65°C.
A higher temperature will inhibit the reaction.
We recommend to control heatblock temperature or switch to a thermocycler.

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.

• 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)