nice question

Article Animal models of nonalcoholic fatty liver disease/nonalcohol...

Jack Son, thanks a lot for attaching this review. In fact, I know this review and read most of its references, particularly HFD models, but basically most of these papers used ficoll or percoll protocol for recoveing IHL and authors didn't mention the process in details.

Hi Mohamed,

In my experience the recovery of small amount of cells after ficoll separation step was not very good. So, you could try to use tubes with polymer gel and sodium heparin (or citrate) for mononuclear cell preparation (CPT). As well as take a look to approach from our colleagues ( Article Phenotypic characterization and functional analysis of human...

I hope the separation media mentioned in article is not ficoll :)

Is it possible to use lipase or maybe some pluronic to "stabilize" lipid drops ( Article Utilizing temperature-sensitive association of Pluronic F-12...

Daniil

Hello Daniil,

Thanks a lot for your comment and for attaching these references.

I read the article'' Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation'', but basically the authors used ficoll for recovering lymphocytes from different solid organ tumors, which the same protocol I used for recovering IHL in my fatty liver model. However, I like the automatic Medimachine™ that they used to mechanically digest the tumor tissues which I think it is a very good option and faster technique than standard mechanical digestion(filter).

Regarding the use of Lipase, thank you so much for suggesting this. In fact, I haven't thought about this before and I think it could be an option to reduce fat content in the liver prior to ficoll separation. However, I don't know much about lipase toxicity on IHL or hepatocytes. Please, if you have any experience about using this enzyme on mice tissues and its safety, I would appreciate if you can share it with me

Hi Mohamed,

Sorry, I don't have experience of lipase using. In theory it should digest fats to gradually to fatty acids + DAG, MAG and glycerin. But how to evacuate so many free FA (albumin is transporter usually)? So, lipase activity possibly will be limited with final products.

One more idea coming from my previous experience of adipose stromal cells isolation. During isolation procedure fat sticks to PP tube as well as to tips. Same to paper - if you will introduce peace of paper to your liquid phase it will accumulate a lot of lipid groplets. So, my idea to try to evacuate most of fat by sterile paper or PP tip and collect layer of mononuclear cells after. During washing steps the residues of fat could be removed or minimized by filtration of suspension via cell stainer.

To find potential solution I have to see protocol. So, I will come to discuss then I will have time.

Have a nice time!

Daniil