Hi everyone,
I am measuring the IL-6 secretion in the J774.A.1 cell line upon the activation with a TLR1/2 ligand.
In my previous studies, everything seemed normal. But recently I have had some results that can't be explained. All the results (considering all my controls) point to the cells themselves. Showing that the cells are behaving differently. Has anybody who worked with J774A.1 macrophage cell line observed any behavior change after a certain passage number?
Thanks
Changes in cellular behavior, including alterations in response to stimuli, can occur in cell lines as they undergo passaging. This phenomenon is commonly referred to as cell line drift or cell line variation.
Here are a few factors that could contribute to cell line drift:
1. Genetic and phenotypic changes: Over time, cells may accumulate genetic mutations or epigenetic modifications, leading to alterations in their phenotype and behavior. These changes can affect their response to stimuli, including the TLR1/2 ligand you are using.
2. Adaptation to culture conditions: Cell lines may adapt and evolve in response to long-term culture conditions, including the composition of the growth medium, culture environment, and subculture techniques. These adaptations can result in changes in cellular behavior and response to stimuli.
3. Contamination: Changes in cellular behavior could be due to contamination with other cell lines or microorganisms. Regularly testing your cell cultures for contamination and maintaining good aseptic techniques are important to minimize this possibility.
4. Passage number: As you mentioned, changes in cellular behavior can sometimes be associated with the passage number. Cell lines, including J774.A.1, may exhibit alterations in growth rates, differentiation potential, or response to stimuli as they are subcultured over multiple passages. Therefore, it is recommended to monitor cell behavior and compare results across different passage numbers to ensure consistency in your experiments.
To address these issues and minimize the impact of cell line drift, it is good practice to periodically validate your cell line using appropriate quality control measures. These may include confirming the expression of relevant markers, comparing response patterns to known stimuli, and periodically reestablishing the cell line from low passage frozen stocks.
If you are observing consistent changes in cellular behavior in your J774.A.1 cell line, it may be beneficial to consult the scientific literature or reach out to researchers who have experience working with this particular cell line. They may be able to provide insights or suggestions based on their own observations or experiences.
best..
Farbod Bahreini,
Passages can change the phenotypes of cells, however cells lines are pretty stable and unless you changed something in the medium they should stay rather stable. If passage induces a phenotypical changes it will be more gradual than drastic. If a drastic change occurs the first thing I would do is test my medium and cells for mycoplasma infection. Unlike bacterial infection the cells won't die (they will, but at a much later stage), the medium doesn't becomes cloudy (until you reach a extremely advanced infection level) and you won't see them even at x40 or more. However they will drastically change the behaviour of your cells, in term of growth (drastic change in growth curve, if you monitor it) and metabolism.
Test treatments and prophylactics exist, they are very good; example:
https://www.invivogen.com/mycostrip
This one detects the 6 most common species of mycoplasma (M. orale, M. hyorhinis, M. arginini, M. fermentans, M. hominis, and A. laidlawii) which account for most of the lab contaminations.
As a note, the first ever paper retracted from Nature Methods was due to mycoplasma contamination of the cell lines (retracted 9 years after publication, nobody could duplicate their findings):
Article Marker-independent identification of glioma-initiating cells
Here they explain why the retractation:
https://blogs.nature.com/methagora/2013/09/retraction_resulting_from_cell_line_contamination.html
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