I'm starting a new project and being new on the field of inflammation, I'd like to know if there are protocols for cultivating human monocyte in vitro and if it is possible to expand and cultivate it. Can someone please point me to some relevant paper? (from a quick research I've found a lot of material).
Hi Denis..thanks for your answer..at first I do not want to differentiate, but just to have monocyte culture..differentiation is later (when I will decide which kind)...
Hi Fulvio, my previous group have cultured primary human monocytes by:
1) Taking human blood
2) Separation of PBMCs by density gradient centrifugation (such as Ficol-paque)
3) Isolation of monocytes by MACs beads: http://www.miltenyibiotec.com/en/PG_58_47_Monocyte_Isolation_Kit_II.aspx OR by allowing the monocytes to adhere to your tissue culture flask/dish/well of choice for 2-4 hours in the incubator, then removing the non-adherent (non-monocyte) cells by washing.
Then culture these cells overnight in fully supplemented DMEM in a 37oC 5% CO2
For macrophage differentiation activate with LPS for 6 hours before use. Protocol in this paper: http://www.jimmunol.org/content/180/12/8410.long
As Denis says, for primary monocytes you will want to use them ideally no more than 24-36 hours after isolation. For longer studies you would need to differentiate them, or if you want to work on the subsequent macrophages there are also a number of macropahge cell lines such as THP-1, RAW and J774 cells are available.
Hi,
I know this a quite old discussion, but this could means that I you had progressed on this topic.
I'm also interested in human monocytes immunophenotype. Is any primary monocyte culture protocol published? And do you also know a specific paper analyzing the differences between fresh isolated and primary cultured monocyte?
Thanks a lot!
I'd be interested in a solution, too. I do not care about some degree of differentiation towards a macrophage phenotype.
Hi Fulvio, in our lab we separate PBMCs by density gradient centrifugation and then for the isolation of primary monocytes we use Human Monocytes enrichment kit:
https://www.stemcell.com/products/easysep-human-monocyte-enrichment-kit-without-cd16-depletion.html
With this kit you have both CD14+ and CD16+ monocytes so basically you can work with the three subsets of monocytes: Classical, intermediate and non classical.
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