I want to isolate and purify Kupffer cells from human with MACS CD14 magnetic beads. Next, we plan to stimulate isolated Kupffer cells with LPS.
My question is whether cd14 beads will influence the effect of LPS on Kupffer cells isolated, since CD14 is the co-receptor of LPS.
Thanks a lot!
I try that before. But the best protocol is by liver perfussion and adhesion. Check
Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
González HE, Eugenín EA, Garcés G, Solís N, Pizarro M, Accatino L, Sáez JC.
Am J Physiol Gastrointest Liver Physiol. 2002 Jun;282(6):G991-G1001.
Shujun -
I agree with Eliseo. If it's possible, avoid engaging CD14 with a particle given your intended downstream use of the Kupffer cells. There are numerous published protocols for isolation based on selective adhesion such as Eliseo's that will likely give you plenty of cells to work with and that won't tie up a critical surface protein in the LPS recognition system.
Dear John and Eliseo
Thank you for your kind suggestions.
We met some problems especially purity (contain many fibroblast) with gradient centrifuge and adhesion method. Our planned experiments need hight purity of macrophage, so we choose CD14 beads to purify non-parenchymal cells suspension. Now our results show these Kupffer cells isolated by CD14 beads still work with LPS treatment. Of course some influence we don't know might have already happened. Maybe we can use CD68 beads to purify.
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