Hello to all,
Lately the HEK293T cell line I am using does not adhere very well to T75 and T175 flask. I wanted to analyse possible factors and conditions that could affect adhesion (trypsin concentration, medium...) and I wanted to ask you, if you know of any way to quantify adhesion. Maybe by measuring the cells in suspension, or give some kind of shock or vortex to the T flask and do a count to see how they have adhered and withstood the impact... I don't know.
I would appreciate your ideas.
Thank you very much,
Ismael
Hello,
The HEK293T cells attach poorly to regular uncoated flask. Coated flask/dish will help in culturing HEK293T cells.
Attachment of cells to the substratum requires interaction of healthy cells with a range of adherent protein molecules present in the serum and supplements added to the culture medium.
Cells fail to attach or show poor attachment to the substratum under sub-optimal growth conditions. Failure to attach to the substratum could also be due to environmental stress caused because of contamination, improper freezing or thawing of cells, using cells with higher passage number, temperature fluctuation in the incubator.
Also, when cells are over trypsinized and when they are subcultured at over confluency, they attach poorly to the surface of the flask.
You just need to know these points whenever you carry out cell culture activities.
I hope this will be helpful.
Good Luck.
Hello,
Several factors can affect cell adhesion in the culture flask.
As HEK-293 is normal embryonic kidney cells, it has slow growth rate than the cancerous ones.
The adherence of cells depends on
1. Concentration of FBS in the media (as it helps in growth). During thawing, 20% FBS is recommended for better growth and speedy adherence.
2. pH of the media. If the media kept for long the pH changes, can cause less adherence.
3. Temperature of the atmosphere and the media while thawing. If the media is chilled, the cells go under stress and can loss adherence property partially.
4. Fluctuations in partial concentration of CO2 chamber.
5. Improper freezing. If the temperature decrease is not maintained properly (first 24hrs in -20 then in -80°c).
6. Passage number also makes them resistant and non adherent to the basement.
And last but not the least trypsin concentration as you already told.
When I grow HEK in laboratory, it takes almost 3-4 days to get 70% confluent.
Thank you,
thank you very much for your reply Esha Sarkar . I notice that when I do the cell passages, when I wash with PBS a large number of cells detach and I would like to minimise this as much as possible.
Dear Ismael,
That should not be happening as PBS is a buffer. But in case if the pH alters, or if the PBS is kept for longer and vigorous washing can cause cell detachments in rare cases.
Rather than washing with PBS, you can try washing the flask with normal media (with 10% FBS). Gently pipette out the washing media and add the fresh one again.
If still the minute contamination stays you can wash with trypsin+ media (1:3). Simply rinse and remove it. For longer stay this will also cause cell detachment. Though this is the ultimate step. Try to avoid trypsin as much as possible.
Thanks,
- Badges
- Science topic
- Similar topics
- Comment