Hello,

Most of the information you've provided aligns with the standard procedures for working with cell lines. However, I'd like to elaborate on my approach when working with HepG2 cells:

Opt for a T-25 flask when reviving cells, taking into consideration the quantity of frozen cells available. Typically, the T-25 flask is suitable.

I traditionally thaw vials, combine them with complete medium at 37 degrees Celsius, and then subject them to a five-minute centrifugation. Following the removal of the supernatant, I resuspend the cells in media before plating. It's worth noting that directly plating without centrifugation can lead to an accumulation of debris from dead cells the next day, although cells can still be revived without centrifugation.

For freezing cells, I recommend using a solution with 5% DMSO. The term "complete media" inherently includes FBS. You can choose to either use complete media for storage or opt for a solution with 90% FBS. In my practice, I prefer using complete media, which includes 5% DMSO and 10% FBS.

Feel free to ask if you have any specific questions or if there's anything else you'd like to discuss regarding cell culture methods

Thank you for your help. I appreciate.

Once I made the revival, some protocol said the complete culture media should supplement first with 20%FBS then after 24 hours change to the media that supplement with 10 %FBS, from your experience is that correct?

I always use 10% FBS, for both reviving and freezing. Different labs have their standardized protocol, you can try both and see what works best for you.

Hello Mnor Faleh

Q1. When we revive the cells with the flask size, it is better to use a 75 cm2 culture flask or a T- 25 cm2 culture flask?

Whenever you revive the frozen cells use a smaller size flask such as T-25 flask because thawing is a stressful process and there are chances that the cell viability may reduce during this process. So, by using a smaller surface area, the cells would be able to be near each other and communicate with one another in a much better way resulting in speedy recovery from the thawing process.

Q2. Some protocol says when you want to revive the cell, transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium 10% EMEM media, and spin at approximately 125 x g for 5 to 7 minutes, then discard the supernatant and resuspend cell pellet with the recommended complete medium, then dispense into a 75 cm2 culture flask contains 15- 20 ml of 10 % of EMEM the complete growth medium, while other protocol says take the entire content of the vial and aliquot into T-25 flask without centrifugation step? Is it butter to centrifuge or not?

Yes, for less sturdy cells which may not be able to withstand centrifugation, you may place the entire content of the vial into T-25 flask without the centrifugation step. The next day, when the cells have attached to the substratum, you may change the growth media to get rid of DMSO present in the freezing medium. However, for HepG2 cells, you may follow the protocol namely, transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium 10% EMEM media, and spin at approximately 125 x g for 5 to 7 minutes, then discard the supernatant and resuspend cell pellet _ _ _ _ _etc., except for the last step namely, dispense into a T-25 cm^2 culture flask containing 10 ml complete growth medium (EMEM + 10% FBS).

Q3. For subculturing, what is the best subculturing ratio to avoid confluency throughout the weekend?

Usually for HepG2, the sub cultivation ratio of 1:4 to 1:6 is recommended. For your requirement as per your question use 1:5 ratio.

Q4. For cell Freezing, what is the best percentage for the DMSO is it 5% or 10%?

Use 5% DMSO.

Q4. Some protocols say 90% of FCS and other says 5% with complete culture media, so which is better FCS or complete culture media? If it is better to use complete culture media, should the media be supported with 10% FBS?

Just use complete growth media which contains 10% FBS and supplement it with 5% DMSO.

Q5. Some protocol said the complete culture media should supplement first with 20%FBS then after 24 hours change to the media that supplement with 10 %FBS, from your experience is that correct?

Yes, whenever you revive the cells, use 20% FBS in growth media for at least 2 passages after thawing so that the cells fully recover from the freezing and thawing stressful conditions. Return to 10% FBS for the subsequent passages.

Good Luck!

Thanks all for helping, I really appreciate