Currently a student new to cell culture techniques.
Whenever I see reagents in protocols I don't know what most of them mean:
Why Dulbecco’s modification of Eagle’s Basal Medium (DMEM) is used for this cell?
Why cells are grown at 5% CO2?
Why is EDTA added to trypsin?
Why fibronectin is used instead of Laminin in PBS?
I see all these culturing protocols and there's no mention as to why a reagent is added and I cannot find any material for stuff like this
There are many good animal cell and tissue culture books, but this is a good place to start: Chapter Animal tissue culture principles and applications
Dear Kahan Bhadani
You can refer to Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition By R. Ian Freshney.
Please refer to the link below for more details on the contents of the book.
Book Culture of Animal Cells: A Manual of Basic Technique and Spe...
I have read this book when I first started my work in cell culture. It was of great help. You will never get all the information in a single book. You will have to do a lot of reading and have discussions with your senior colleagues who have experience in cell culture. You will learn many things in cell culture, especially (as you mentioned why a reagent is added) as you start working in this area of research under the guidance of seniors. You will not come across any detailed information in any text book. It comes with experience after you work in cell culture. Nevertheless, you will definitely find the book written by R. Ian Freshney very useful.
Good Luck.
you can try give a look here
https://cellculture.altervista.org/?doing_wp_cron=1642544171.8824760913848876953125
To answer some your questions directly:
- DMEM is traditionally used for adherent cells but you can easily adopt cells to different formulations.
- CO2 it the counterpart of the medium pH buffer. The optimal concertation depends on the concentration of sodium bicarbonate in your medium. 5% CO2 is the commonly used compromise but defiantly not the optimum for all cells and media. Check-out the CO2 calculator at www.cellseeker.org/cellcalc/
- EDTA complexes bivalent ion (Ca2+, Mg2+) which are required for adherence proteins to work and is thereby supporting the detachment. This is also why your should use PBS w/o Ca2+ and Mg2+ to dilute trypsin/EDTA.
- Fibronectin is used to coat TC flasks to improve the attachment of cells.
If you are working with cells a lot you should check-out Cellseeker (https://www.cellseeker.org/). This is an easy and free to use app to organize and inventory your cell collection online.
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