DNA Fluorochrome Staining Assay with Indicator Cell Line

M-150 Procedural Summary

DNA fluorochrome staining assay with sample inoculation on an indicator cell line. This approach enhances the assay sensitivity by reducing background from genetically unstable cell lines (e.g. hybridomas, etc.) and amplifies the titer of mycoplasma contaminants. Developed for the detection of mycoplasma contamination in cell cultures and related samples, this test provides reliable and timely results.

  • The indirect method with DNA fluorochrome staining involves the inoculation of 1.0 mL of sample volume into a mycoplasma free indicator cell line (Vero). 
  • Cell cultures are incubated at 36°C± 1°C with 5% CO2 and 95% air for a period of 3 to 5 days.
  • Following incubation, the slide culture is fixed and stained with a DNA-binding fluorochrome (Hoechst 33258). The extranuclear fluorescence staining pattern is then interpreted with UV-fluorescent microscopy up to 600x magnification.
  • Advantages of this assay include the enhancement of sensitivity and specificity by reducing background from genetically unstable cell lines (e.g. hybridomas, etc.), amplification of mycoplasma titers and detection of "non-cultivable" organisms.
  • Appropriate positive (< 100 CFU) and negative controls are processed with each sample.
  • GMP test
  • Total testing time is 4-5 days.

ADHERENT Cells:

Grow adherent cell culture in a T-25 flask with antibiotic free media. When the cell culture is nearly confluent, scrape and aliquot 2 mL to 10 mL of the cell suspension. Place sample aliquot into a sterile screw cap 15 mL polypropylene centrifuge tube for shipment under frozen or ambient conditions. Alternatively, sample may be submitted in a T-25 flask ambient. In this situation, simply add fresh antibiotic free media to the vessel prior to shipping. Leave sufficient room in the neck of the flask to allow for expansion and to prevent leakage. Samples should be cryopreservative free and have undergone at least 3 passages in antibiotic free media prior to submission for mycoplasma testing to achieve the highest level of assay sensitivity. It is recommended that a back-up sample be retained and stored at -60oC or colder.

NONADHERENT Cells:

Take 2 mL to 10 mL of non-adherent cell suspension and place in a sterile screw cap 15 mL polypropylene centrifuge tube. Samples should be cryopreservative free and have undergone at least 3 passages in antibiotic free media prior to submission for mycoplasma testing. It is recommended that a back-up sample be retained and stored at -60oC or colder.

Shipping Instructions:

Ship Frozen on dry ice or ship under ambient conditions. Bionique Testing Laboratories® recommends frozen shipment on dry ice. A 2 mL to 10 mL sample aliquot should be frozen in a sterile 15 mL polypropylene screw cap centrifuge tube and shipped overnight in an insulated container with sufficient dry ice to keep frozen for a period of 48 hours. If shipping sample under ambient conditions, a nearly confluent T-25 flask or 2 mL - 10 mL sample aliquot in a 15 mL polypropylene screw cap centrifuge tube should be used. Place the sample vessel inside a plastic bag in a crush proof package and ship overnight.

Be aware that shipping under ambient conditions may lead to sample compromise due to leakage, cell overgrowth, cell death, exposure to cyclic extremes in temperature, etc.

Please view our general sample submission instructions page for details on how to submit your sample(s) online.



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Total Testing Time: 4-5 days
Sample Requirements: Minimum Test Volume = 2 mL

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