Cell Culture Q&A: Min. Cell Amount, Trypsin Inhib., pH & Osmolarity

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Determining the minimum cell amount for a specific cell line to enter log phase growth involves plating different dilutions to identify the threshold for normal growth. If using medium with serum that contains a trypsin inhibitor, washing cells with PBS before adding trypsin is recommended for effective dislodging. HCO3- is essential in the medium as it buffers pH and can serve as an energy source for cells. Maintaining a pH between 7.0 and 7.4 is crucial, along with osmolarity levels similar to physiological conditions, around 150 millimolar. Incubating cells in a CO2 environment mimics in vivo conditions, balancing oxygen and CO2 levels for optimal growth.
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# How do I know the minimum of cells for a specific cell line that give a quick entry into log phase growth?

#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?

#Should I always have HCO3- in the medium and what for?

# Should pH always be between 7.0 to 7.4 in the medium? What about the osmolarity?

Hope for any ideas.

Thanks.
 
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sobored said:
# How do I know the minimum of cells for a specific cell line that give a quick entry into log phase growth?

Plate different dilutions of your cell line and see were the cut off point is, usually there's a treshold were the cells are suddenly to sparsely plated to give rise to normal growth.

sobored said:
#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?

Trypsin, just wash your cells once with PBS be4 adding the trypsin and you;ll be fine

sobored said:
#Should I always have HCO3- in the medium and what for?

Its a buffer to keep the CO2 from the incubator to change the pH of the culture medium, it can also serve as an energy source for your cells

sobored said:
# Should pH always be between 7.0 to 7.4 in the medium? What about the osmolarity?

Yes your cells won't have it any other way unless there bacterial :wink: osmolarity has to be the same as in the body like PBS, that's around 150 milimolars if I am not mistaken
 
Thank you. :smile:
 
Sho'Nuff said:
Its a buffer to keep the CO2 from the incubator to change the pH of the culture medium, it can also serve as an energy source for your cells


I forgot to ask why should we incubate our cells in the CO2 incubator when we know that the CO2 will change the pH of our medium? Won't this create more problems?

Any ideas?

Thanks.
 
We try to mimick the inside of the body ad good as we can in an incubator to obtain data that is more relevant to the situation in vivo. That means less oxygen (which is a stress factor to the cells) and more CO2, cells won't grow without it. CO2 is also part of the buffering system of the culture medium. Its a delicate balance...
 
I see. :smile: :cool:
 
sobored said:
#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?
It is good to know that fcs (fetal calfs serum) contains a trypsin inhibitor. I wash my cells with some PBS and then do the digestion in some PBS with trypsin. Just don't leave the PBS on the cells too long: they don't like that much.

You can also dislodge your cells with a plastic scraper when cells are not sensitive to trypsin digestion.
 

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