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. 2021 Sep 14;2(4):100831.
doi: 10.1016/j.xpro.2021.100831. eCollection 2021 Dec 17.

Isolation of mouse Kupffer cells for phenotypic and functional studies

Francesco Andreata  1 , Camille Blériot  2 , Pietro Di Lucia  1 , Giorgia De Simone  1   3 , Valeria Fumagalli  1   3 , Xenia Ficht  1 , Cristian Gabriel Beccaria  1 , Mirela Kuka  1   3 , Florent Ginhoux  2   4   5 , Matteo Iannacone  1   3   6
Affiliations

Isolation of mouse Kupffer cells for phenotypic and functional studies

Francesco Andreata et al. STAR Protoc. .

Abstract

Here, we provide detailed protocols for the isolation of mouse Kupffer cells - the liver-resident macrophages - for phenotypic (e.g., via flow cytometry, mass cytometry, or RNA-sequencing) analyses or for functional experiments involving cell culture. The procedures presented can be adapted for the isolation of other hepatic cell populations. For complete details on the use and execution of this protocol, please refer to De Simone et al. (2021).

Keywords: Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Single Cell.

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Conflict of interest statement

M.I. participates in advisory boards/consultancies for Gilead Sciences, Roche, Third Rock Ventures, Amgen, Allovir. M.I. is an inventor on patents filed, owned, and managed by San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Telethon Foundation on technology related to work discussed in this manuscript (WO2020/016434, WO2020/016427, WO2020/030781, WO2020/234483, EU patent applications n. 19211249.8 and n 20156716.1, and UK patent application n. 1907493.9). F.G. is a member of the Immunity advisory board.

Figures

None
Graphical abstract
Figure 1
Figure 1
Surgical preparation and harvesting of the liver (A and B) (A) Euthanized mouse is sanitized with 70% ethanol and the peritoneal cavity is exposed (B). (C) The intestine is pushed sideward in order to expose the portal vein and the vena cava. (D and E) (D) Liver is perfused through the vena cava. After 2 mL of perfusion the liver vasculature becomes engorged (E); at this moment the portal vein is cut. (F and G)(F) Liver is perfused with 10 mL of PBS and the organ clears immediately turning into a light brown color (G). (H–J) (H) The gallbladder is removed, and the hepatic ligaments are cut (I) before the complete harvest of the liver (J).
Figure 2
Figure 2
Configuration of the equipment for the in situ digestion of the liver
Figure 3
Figure 3
Different strategies to gate live cells Representative dot plots of LNPC preparation in which live cells have been gated with DAPI staining (A) or with fixable Live/Dead staining (B). In separate samples, LNPCs have been mixed in 1:1 ratio with LNPCs placed at 70°C for 5 min to show actual dead cell population.
Figure 4
Figure 4
Gating strategy for Kupffer cell and LSEC identification in LNPC samples isolated from C57BL/6 mice Representative dot plots of LNPC preparation obtained with the quick ex vivo digestion protocol (A) or with the in situ digestion protocol (B). Single, live cells can be divided in LSEC (CD45-, CD31+) and in hepatic leukocytes (which are CD45+). Once discarded the lineage positive cells (CD3+, CD19+, CD49+, Ly6G+), the F4/80+CD11bint macrophage population is pre-gated to further distinguish capsular macrophages (TIM4-, I-A/I-E+) from Kupffer cells (TIM4+, I-A/I-Eint). Kupffer cells (KCs) can be divided in KC1 (CD206-, ESAM-) and in KC2 (CD206+, ESAM+) subpopulations.
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References

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