Thank you very much, Harald. I’ll look into that. Can I ask if you have any experience working with HepG2 cells? What do you think is the optimal cell density for 6-well, 24-well, and 96-well plates? Thanks again

Hi Murinah

HepG2 tend to be difficult regarding the type of plastic and also if your culture ages. Usually, if you want to improve cellular adhesion try 0.4% collagen, just a short incubation of five minutes before adding the media could improve adherence. HepG2 do not adhere to glass, so be carefull.

Most people encounter problems with excessive time of trypsin, because they tend to adhere to each other and then they do not adhere to the plastic because of membrane damage. Some trypsin solutions have edta, for my cells the one from gibco (25300054) works ok, but I've encountered other with higher percentages of edta that inhibit adhesion after some passages.

Hope it helps

Hi Mónica Almeida,

Thank you so much for the helpful information!

1- May I ask what plastic brand you use?

2- Regarding the incubation in 0.4% collagen, if you dissolve 0.4 grams of collagen in 100 mL of a buffer like 1X PBS, how would you apply it? Do you apply the collagen solution directly to the surface, or do you mix it with the cells?

3- Also, how much of the 0.4% collagen solution do you typically use?

I’m currently struggling with these cell lines, and I’m really grateful to connect with someone who can share such valuable insights. If you could please follow me in Researchgate so I can send you some images to hear your opinion about these cells.

Thanks a lot again.

Munirah Faleh I use both thermofisher delta nucleon and a new brand which is Nest and also orange scientific. You can just add enough collagen to cover the surface and let it rest for 5 min inside the flow chamber and then just collect it again and add the media directly, without washing the plate. if you have collagen in powder that is the correct dose, otherwise just buy rat collagen solution from any vendor, already in liquid form. it is ok, to use a ore concentrated form, but since collagen tends to be expensive, we can diminish it's use up to 0.1% máximum, otherwise will not have effect.

But I would check the type o trypsin your using to passage the cells, because that almost always the main culprit of problems regarding low attachment.

Best of luck

Thanks, Mónica! I've attached the trypsin type for your reference.

1- Do you think coating the plate with collagen would help form a good cell monolayer? Or do you use any other techniques for this purpose? Forming cell monolayer is very important as i will do Knockout on these cell lines and virus infection

2- Also, have you tried seeding cells in 96-well and 24-well plates? What cell density worked best for you?

I apologize for the detailed questions, but I would appreciate your insights. Thanks again!

Hi Munirah Faleh that is too much trypsin, you can use 0.05% trypsin and it should be enough. For this percentage, probably 1 or 2 minutes of incubation should be enough, more than that it will damage your membranes. You can try to diluted a bit and then let it incubate for 4 min and check if they attach better.

For monolayer formation you only need to be thorough when resuspending the pellet after centrifugation. Add a step for resuspension with a micropipette of 1000 ul in 2 ml of medium, before adding the final amount of media and that will help to have smaller groups of cells when platting, and that will diminish the number of islands you get when they are growing. Collagen will always help to spred the cells, if necessary just add it previously and you should be ok.

Cell density is always dependent on the application you are seeking and time-points you are going to analyse, just use the values of reference that are available (usually based in HeLa) and adjust for your need.

https://www.thermofisher.com/pt/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html

Hope it helps

Best

Mónica