Technical library

Regular mycoplasma testing is an important part of good cell culture practice because contamination may remain unnoticed and can affect cell growth, morphology, and experimental results.
Recommended testing frequency:
The appropriate testing frequency depends on the type of cells and how long they are maintained in culture:
- Continuous or long-term cell lines: Test regularly throughout cultivation. Monthly testing is a practical routine for continuously maintained cultures.
- Primary human cells: These cells have a finite lifespan and are generally cultured for a limited number of population doublings. Therefore, monthly testing may not always be necessary for short-term experiments. We recommend additional testing at a relevant experimental checkpoint, particularly before important experiments or before cryopreserving expanded cells, or whenever contamination is suspected.
- Newly introduced cultures: Consider testing cells when they are first introduced into your laboratory, especially if their mycoplasma status is unknown. All PromoCell cell lots are tested for the absence of mycoplasma as part of quality control before release. For PromoCell primary cells, additional testing immediately after receipt is therefore generally not necessary. 
- Suspected contamination: Test immediately if you observe unexpected changes in cell growth, morphology, or culture behavior.

Good post-thaw viability does not necessarily mean that primary cells will immediately proliferate. If the cells survive thawing and attach but show little or no growth, first check the culture conditions and handling steps.

Quick checks:
Growth medium and supplements
Make sure you are using the recommended growth medium for your specific cell type and that all required supplements have been added correctly. The recommended media are important for achieving the expected proliferation potential.
Thawing procedure
Review the thawing procedure. For anchorage-dependent primary cells, the established protocol specifies thawing followed by plating at the recommended seeding density. Depending on the protocol, cells can be directly plated or centrifuged before seeding.
Seeding density
Check that the cells were seeded at the density recommended in the cell-specific Instruction Manual. The established thawing protocol specifically requires plating cells at their recommended seeding density.
Cell attachment and morphology
Examine the culture microscopically and monitor whether the cells attach and develop the morphology expected for the respective primary cell type.
Post-thaw lag phase and population doubling time (PDT)
Keep in mind that some primary cells or cell types may have a prolonged lag phase after thawing before active proliferation becomes apparent. Cells with a longer population doubling time (PDT) may also initially appear to grow very slowly, even when the culture is developing normally. Check the lot-specific PDT stated in the Certificate of Analysis (CoA) when assessing whether the observed growth rate is within the expected range.
First medium change
Avoid changing the medium too early unless the cell-specific protocol specifies otherwise. For cells centrifuged before plating, the general published thawing protocol specifies changing the medium no earlier than 24 hours after seeding. Some cell types have different requirements.
Cell-type-specific requirements
Always check the Instruction Manual for the respective cell type, as thawing, seeding, coating, medium change, and culture requirements can differ.

If proliferation remains impaired after the recommended culture conditions and the expected PDT have been considered, please contact our Scientific Support team for assistance with troubleshooting.