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Murine Liver Myeloid Cell Isolation Protocol
鼠肝髓样细胞分离法   

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Abstract

In homeostasis, the liver is critical for the metabolism of nutrients including sugars, lipids, proteins and iron, for the clearance of toxins, and to induce immune tolerance to gut-derived antigens. These functions predispose the liver to infection by blood-borne pathogens, and to a variety of diseases ranging from toxin and medication-induced disorders (CCl4, acetaminophen) to metabolic disorders (steatohepatitis, alcoholic liver disease, biliary obstruction, cholestasis) or autoimmunity. Chronic liver injury often progresses to life threatening fibrosis and can end in liver cirrhosis and hepatocellular carcinoma (Pellicoro et al., 2014).
The liver contains parenchymal cells or hepatocytes that make up the majority of hepatic cells. It also contains non-parenchymal structural cells such as sinusoidal endothelial cells and a large number of non-parenchymal innate immune cells, mainly monocytes, neutrophils, macrophages, DCs, NK and NKT cells that can trigger an adaptive immune response in the case of infections or other pathogenic insults (Jenne and Kubes, 2013). How this immune response is regulated determines the extent of acute and chronic liver injury (Stijlemans et al., 2014). In this context, liver macrophages have been demonstrated to play central but divergent (from initiating to resolving) functions in liver injury (Sica et al., 2014). It has become clear in the last years that hepatic macrophages consist of two classes, tissue-resident macrophages, the Kupffer cells (KCs) originating from yolk sac/fetal liver progenitors and tissue-infiltrating macrophages originating from bone marrow-derived Ly6CHi monocytes (Jinhoux and Jung, 2014; Tacke and Zimmerman, 2014). Distinguishing the activities of KCs from those of monocyte-derived macrophages during liver injury or repair is currently a frontline research topic in the macrophage field. Indeed, considering that clinical management of liver failure remains problematic, a better understanding of the immune mechanisms regulating liver injury is expected to allow the development of new therapeutic modalities. Here, we describe an isolation technique for liver non-parenchymal polymorphonuclear (PMN) and mononuclear myeloid cells permitting their molecular and functional characterization.

Keywords: Myeloid Cell(髓系细胞), Kupffer Cell (KC)(Kupffer Cell(KC)), Monocyte-derived macrophages(单核细胞源性巨噬细胞), Polymorphonuclear (PMN) myeloid cell(多形核粒细胞(PMN)), Liver injury(肝损伤)

Materials and Reagents

  1. 7-8 weeks old female C57Black/6 mice (Janvier Labs)
  2. RPMI-1640 medium (RPMI) (Life Technologies, catalog number: 52400-041 )
  3. Collagenase Type III (Worthington Biochemical, catalog number: LS004180 )
  4. DNase I (Roche Diagnostics, catalog number: 04536282001 )
  5. Heparin (sodium salt from porcine intestinal mucosa) (Sigma-Aldrich, catalog number H3393-1MU )
  6. Hank’s buffered salt solution (HBSS) without calcium or magnesium or phenol red (Life Technologies, Gibco®, catalog number: 14175-053 )
  7. NH4Cl (Merck KGaA, catalog number: 01145.0500 )
  8. KHCO3 (Merck KGaA, catalog number: 04854.0500 )
  9. EDTA (Duchefa Biochemie, catalog number: E0511.1000 )
  10. HCl (37% stock solution) (Merck KGaA, catalog number: 1.00317.1000 )
  11. Fetal bovine serum (FBS) (BiowhittakerTM/Lonza, catalog number: DE14-801F )
  12. LymphoprepTM (Axis-shield, catalog number: 1114547 )
  13. PercollTM (GE Healthcare, catalog number: 17-0891-01 )
  14. Purified CD16/CD32 (Fc-Block) (clone 2.4G2) (BD Biosciences, catalog number: 553142 )
  15. PE-Cy7-conjugated anti-CD11b antibody (clone M1/70) (BD Biosciences, catalog number: 552850 )
  16. AF647-conjugated anti-Ly6C antibody (clone ER-MP20) (Serotec, catalog number: MCA2389A647 )
  17. PerCP-Cy5.5-conjugated anti-I-A/I-E (MHC-II) antibody (clone M5/114.15.2) (Biolegend, catalog number: 107626 )
  18. FITC-conjugated anti-Ly6G antibody (clone 1A8) (BD Biosciences, catalog number: 551460 )
  19. APC-Cy7-conjugated CD45 antibody (clone 30-F11) (BD Biosciences, catalog number: 103116 )
  20. PE-conjugated F4/80 antibody (clone CI: A3-1) (AbD Serotec, catalog number: MCA497PET )
  21. Trypan blue (BDH Chemicals, catalog number: 34078 )
  22. NaCl (Thermo Fisher Scientific, catalog number: 10428420 )
  23. KH2PO4 (Merck KGaA, catalog number: 1.04873.1000 )
  24. Na2HPO4.2H2O (Merck KGaA, catalog number: 1.06580.1000 )
  25. L-glutamine (Sigma-Aldrich, catalog number: G8540-100G )
  26. Penicillin (Life Technologies, Gibco®, catalog number: 15140-122 )
  27. Streptomycin (Life Technologies, Gibco®, catalog number: 15140-122)
  28. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  29. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360-039 )
  30. Non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-035 )
  31. Liver digestion medium (see Recipes)
  32. Phosphate buffered saline (PBS) (see Recipes)
  33. 33% Percoll working solution (see Recipes)
  34. Erythrocyte lysis buffer (see Recipes)
  35. MACS buffer (see Recipes)
  36. Cell suspension medium (see Recipes)
  37. Blocking medium (see Recipes)
  38. Complete medium (see Recipes)
  39. Trypan blue working solution (see Recipes)

Equipment

  1. Polyester filters cut in 10 x 10 cm squares, thread diameter 70 μm (Spectrumlabs, catalog number: 146490 )
  2. 10 ml syringes (Omnifix, catalog number: 473203 )
  3. BD Falcon 50 ml polypropylene tubes (BD Biosciences, catalog number: 2070 )
  4. BD Falcon 15 ml polypropylene tubes (BD Biosciences, catalog number: 2096 )
  5. BD Falcon 5 ml polypropylene round-bottom tube (BD Biosciences, catalog number: 352063 )
  6. Needles (Microlance 22G1 ½, 0.7 * 40 mm) (BD Biosciences, ref: 301000 )
  7. Sterile culture hood
  8. Surgical scissors and forceps
  9. 37°C, 5% CO2 cell culture incubator (Forma Scientific)
  10. Pipettes
  11. Centrifuges (Eppendorf, models: 5810R and 5417C )
  12. Orbital shaker (Belgolabo, model: Julabo type SW-20C ) used at 200 rpm
  13. Light microscope (Olympus, model: CK2 )
  14. Multicolor flow cytometer (BD Biosciences, FACSCantoTM )
  15. GentleMACSTM Dissociator (Miltenyi Biotec, catalog number: 130-093-235 )
  16. GentleMACSTM C-tubes (Miltenyi Biotec, catalog number: 130-093-237 )

Procedure

  1. Preparation of a liver single cell suspension
    1. Sacrifice the mouse using CO2 and restrain it by pinning its paws into a foam surface using syringe needles. Of note, all murine experiments were performed according to the ECPVA guidelines (CETS n° 123) and were approved by the VUB Ethical Committee (Permit Number: 08-220-8). Heparinized blood was taken via cardiac puncture (~1 ml) in order to prevent too much blood contamination when taking the liver. Alternatively, the liver can be perfused in vivo via the portal vein with 10 ml saline. Make a parallel incision from the base of the tail up to the neck along the mouse’s abdomen and to the paws without puncturing the peritoneum. Gently pull back the skin and pin it to the foam surface. Subsequently, open the mouse abdomen and softly move the intestines on the side to get access to the liver using a cotton plug.
    2. Gently take out the liver from the body, without damaging it.
    3. Store the harvested liver in 5 ml RPMI medium in a 50 ml Falcon tube on ice until the digestion procedure.
    4. Put the liver in a GentleMACSTM C-tube and add 5 ml liver digestion medium. Subsequently, cut the liver in small pieces (1-1.5 mm) using scissors (Figure 1A/B).
    5. Homogenize the liver using the GentleMACSTM Dissociator (Figure 1C) using 2 times program mLiver_01_03 (16 sec) at room temperature. Subsequently, incubate at 37 °C for 20-30 min while shaking in a water bath to allow digestion of the tissue.
    6. Homogenize the liver suspension once more using the GentleMACSTM Dissociator program mLiver_02_03 (25 sec) at room temperature (Figure 1D). Finally, add 5 ml of blocking medium to stop the liver digestion.
    7. In order to measure the cytokine content within whole liver, collect 0.5 ml of the solution in a 1.5 ml Eppendorf tube and centrifuge it at 10,625 x g for 8 min at room temperature. Subsequently, collect the supernatant and store at -20 °C till needed.
    8. Filter the remaining liver suspension through a 70-µm sterile nylon gauze into a sterile 50 ml conical tube. Wash the GentleMACSTM C-tube with an additional 20 ml of blocking solution and rinse the filter once more with this.
    9. Centrifuge the 50 ml tubes at 450 x g for 8 min at 4 °C and gently discard the supernatant.
    10. Eliminate the red blood cells by resuspending the cell pellet in 5 ml ice-cold erythrocyte lysis buffer and leaving it on ice for 2-3 min.
    11. Neutralize the lysis by adding 25 ml cell suspension medium, and transfer the suspension to a new 50 ml tube through a 70-µm sterile nylon gauze.
    12. Centrifuge the 50 ml tube at 450 x g for 8 min at 4 °C and gently discard the supernatant.
    13. Resuspend the cell pellet in 5 ml cell suspension medium and count the living cells using Trypan blue.
      At this stage, the suspension contains both parenchymal (hepatocytes) and non-parenchymal (including polymorphonuclear cells, monocytes, macrophages, DCs, NK and NKT cells) liver cells. Although this suspension can be analyzed via flow-cytometry, it is advisable to perform additional fractionation steps to allow cell culturing and/or FACS sorting.

  2. Fractionation of liver cells using a Lymphoprep gradient separation
    1. Add slowly 10 ml LymphoprepTM (Lucron Bioproducts) underneath the resuspended liver cell solution using a 10 ml syringe with long needle. Centrifuge at 800 x g for 25 min at 20 °C without acceleration or brake (Figure 1E).
    2. Carefully collect the layer of low-density cells at the interphase containing the non-parenchymal liver cells (enriched in mononuclear myeloid cells as well as T-, B- and NK/NKT-cells). If the interphase is not clearly visible (low amount of cells) you can also collect the upper phase (containing RPMI medium). Take as less as possible of the lower phase containing Lymphoprep (Figure 1E).
    3. Transfer the interphase to a new sterile 15 ml tube and fill to the top with MACS buffer. Centrifuge at 800 x g for 7 min at 20 °C and discard the supernatant. Resuspend the cell pellet in 1-2 ml cell suspension medium, and after counting the cells using Trypan blue, bring at a concentration of 107 cells/ml.
    4. After removal of the Lymphoprep, collect the lower fraction (pellet) containing mainly parenchymal cells (hepatocytes) and polymorphonuclear (PMN) cells that were not retained within the non-parenchymal cell fraction due to their high density characteristics and transfer it to a sterile 15 ml Falcon tube (Figure 1E). Resuspend the cells in a final volume of 15 ml cell suspension medium and centrifuge at 650 x g for 8 min at 20 °C.
    5. Subsequently, in order to remove the Lymphoprep solution perform 1-2 washing steps of 15 ml cell suspension medium. Finally, resuspend the pellet in 3 ml cell suspension medium and, after counting the cells using Trypan blue, bring at a concentration of 107 cells/ml.
      The interphase (non-parenchymal cells) fraction can now be used for FACS analysis/sorting as well as for cell culturing conditions. The bottom fraction containing hepatocytes and remaining non-parenchymal cells (PMN as well as macrophages with higher density) can also be used for flow-cytometric analysis. The latter fraction can be further cleaned-up using a Percoll gradient in order to separate hepatocytes from the remaining non-parenchymal cells. Of note, if hepatocytes are not needed DNase 1 can be added to the digestion medium, resulting in a cleaner sample.

  3. Fractionation of liver cells using a Percoll gradient separation
    1. To separate hepatocytes from the PMN and remaining macrophages, mix 1 vol. (~ 5 ml of the 107/ml working solution) of cell suspension with 1 vol. of isotonic Percoll solution of density 1.07 g/ml (33% working solution). Centrifuge for 30 min at 800 x g at room temperature (Figure 1F). Aspirate hepatocytes, forming a broad band at a density of 1.07-1.09 g/ml, from the gradient and wash free of Percoll by two additional cycles of centrifugation at 800 x g, for 5 min and resuspension in 3 ml cell suspension medium.
    2. After removal of the Percoll, collect the lower cell pellet fraction containing mainly PMN and macrophages that were not retained within the non-parenchymal fraction due to their density characteristics and transfer it to a sterile 15 ml Falcon tube.
    3. Subsequently, centrifuge at 650 x g for 5 min. at 4 °C, resuspend the pellet in 1 ml cell suspension medium, count the cells using Trypan blue and bring them at a concentration of 107 cells/ml.
      The different fractions obtained in sections B and C can be used for flow-cytometric analysis or when resuspended in complete medium for in vitro cell culturing.


      Figure 1. Liver single-cell preparation. A. Liver in a GentleMACSTM C-tube in 5 ml digestion medium. B. Liver in a GentleMACSTM C-tube after cutting with scissors. C. GentleMACSTM Dissociator. D. Liver in a GentleMACSTM C-tube after digestion and homogenization using the GentleMACSTM Dissociator. Subsequently, filter the suspension and lyse RBCs. E. Gradient after centrifugation with a clearly visible interphase (non-parenchymal cells, red arrow) and a pellet (hepatocytes and remaining non-parenchymal cells with higher density). F. Optional: Percoll gradient after centrifugation with a clearly visible upper fraction (hepatocytes, blue arrow) and a pellet (PMN and remaining non-parenchymal cells with higher density).

  4. Flow-cytometric analysis
    1. Transfer 100 µl of a 107 cells/ml stock solution of the different fractions [(interface Lymphoprep (section B), pellet Percoll (section C)] into a 5 ml polypropylene round-bottom tube to prevent sticking of cells. Incubate the cell suspension with 1 µg rat anti-mouse CD16/CD32 FcR-blocking antibody clone (2.4 G2, 1 µg per 106 cells) on ice water for 20 min.
    2. Subsequently, add fluorescently labeled antibodies (0.2 µg per 106 cells) for another 20 min on ice water, protected from exposure to light. Antibodies used are FITC-conjugated Ly6G, PE-conjugated F4/80, APC-conjugated Ly6C, APC-Cy7 conjugated CD45, PE-Cy7-conjugated anti-CD11b and PerCP-Cy5.5-conjugated MHC-II.
    3. Wash by adding 2 ml ice-cold MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatant. Add 100-200 µl cell suspension medium to keep cells alive, transfer the cells into a FACS tube and proceed to the FACSCantoTM.
    4. Analyze the FACS data using FlowJo software. Briefly, after selecting a life gate and single cells (using a FSC-A versus FSC-H profile) select the CD45+ cells in a CD45 versus FSC-A plot (Figure 2, upper and lower panels). Within the CD45+ cells, gate out the PMN based on their CD11bpos Ly6Gpos expression profile and select for the CD11bpos Ly6Gneg expressing cells (Figure 3A). The CD11bneg Ly6Gneg population consists mainly of T-cells, B-cells and NK-cells. Next, plot the CD11bpos Ly6Gneg cells in a Ly6C versus MHC-II plot and subsequently check for F4/80 expression. As such, monocytes as Ly6Chigh MHC-IIneg F4/80low cells, monocyte-derived “immature” macrophages as Ly6Chigh MHC-IIhigh F4/80high cells, resident/mature macrophages (i.e. Kupffer cells/”mature” monocyte-derived macrophage) as Ly6Cneg/low MHC-IIhigh F4/80high cells, patrolling monocytes as Ly6Cneg/low MHC-IIneg F4/80low cells and eosinophils based on their Ly6Cint MHC-IIneg F4/80low expression are identified (Figure 3B). Of note, the identity of eosinophils is confirmed based on their SiglecF expression and high SSC.


      Figure 2. Representative FACS gating strategy for different liver preparations (total liver suspension, Lymphoprep upper fraction, Lymphoprep lower fraction, Percoll lower fraction and Percoll upper fraction). (Upper) Selection of a life gate based on a SSC-A versus FSC-A plot. (Lower) After selection of single cells, select CD45+ cells in a CD45 versus FSC plot.


      Figure 3. Representative gating strategy for myeloid cells within the CD45+ fraction. A. Plot the CD45+ cells (see Figure 2, lower panels) in a Ly6G versus CD11b plot to identify PMN (CD11bpos Ly6Gpos), mononuclear cells (CD11bpos Ly6Gneg) and CD11bneg Ly6Gneg cells (T-cells, NK-cells, B-cells). B. The remaining CD11bpos Ly6Gneg fraction is plotted in a MHC-II versus Ly6C plot to allow identifying monocytes (Ly6ChighMHC-IIneg), monocyte-derived “immature” macrophages (Ly6Chigh MHC-IIhigh), “resident/mature” macrophages (Kupffer cells, Ly6Cneg/low MHC-IIhigh), patrolling monocytes (Ly6Clow/negMHC-IIneg) and eosinophils (EO, Ly6CintMHC-IIneg). C. F4/80 plot of the inflammatory monocytes (black), patrolling monocytes (blue), Monocyte-derived “immature” macrophages (green) and “resident/mature” macrophages (orange).

Recipes

  1. Liver digestion medium
    1 ml Collagenase Type III stock: 6,000U/ml) diluted in 50 ml Hanks' Balanced Salt Solution (HBSS) without calcium or magnesium
    Aliquot the stock solution of Collagenase Type III (1 ml/tube) and freeze at -20 °C
    Optional: If hepatocytes are not needed add 10 Units/ml of DNase I to the medium.
  2. Phosphate buffered saline (PBS)
    8 g/L NaCl
    0.2 g/L KCl
    0.24 g/L KH2PO4
    1.8 g/L Na2HPO4.2H2O
    Add distilled water till 1 L and adjust pH: 7.4 with HCl (stock solution 37%)
  3. 33% Percoll working solution
    Add PBS to 16.5 ml PercollTM stock solution till 50 ml final volume
  4. Erythrocyte lysis buffer
    8.29 g/L NH4Cl
    1 g/L KHCO3
    37.2 mg/L EDTA
    Add distilled water till 1 l and bring at pH 7.2 using HCl
  5. MACS buffer
    HBSS without calcium or magnesium or phenol red
    2% (v/v) heat-inactivated fetal bovine serum (FBS)
    3 mM EDTA
  6. Cell suspension medium
    RPMI
    5% (v/v) heat-inactivated fetal bovine serum (FBS)
  7. Blocking medium
    HBSS without calcium or magnesium or phenol red
    2% (v/v) heat-inactivated FBS
    5 mM EDTA
  8. Complete medium
    Roswell Park Memorial Institute (RPMI)-1640
    10% (v/v) heat-inactivated fetal bovine serum (FBS)
    300 μg/ml L-glutamine
    100 U/ml penicillin
    100 μg/ml streptomycin
    0.02 mM β-mercaptoethanol
    1 mM sodium pyruvate
    1 mM non-essential amino acids
  9. Trypan blue working solution
    Make a 1% v/v Trypan blue solution in PBS and use it in a 1/10 ratio (Cells/Trypan blue)

Acknowledgments

We acknowledge the financial support of the Interuniversity Attraction Pole Program (PAI-IAP N. P7/41, http://www.belspo.be/belspo/iap/index_en.stm) and a grant from the FWO (KaN 1511812N). Benoit Stijlemans is a research fellow supported by the VUB/SRP Targeting inflammation linked to infectious diseases and cancer (Nanobodies for Health). The authors also thank Ella Omasta, Marie-Therese Detobel, Maria Slazak, Victor Orimoloye and Nadia Abou for technical assistance. We also would like to thank Dr. Carl De Trez for his constructive discussions.

References

  1. Ginhoux, F. and Jung, S. (2014). Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat Rev Immunol 14(6): 392-404.
  2. Jenne, C. N. and Kubes, P. (2013). Immune surveillance by the liver. Nat Immunol 14(10): 996-1006.
  3. Pellicoro, A., Ramachandran, P., Iredale, J. P. and Fallowfield, J. A. (2014). Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 14(3): 181-194.
  4. Sica, A., Invernizzi, P. and Mantovani, A. (2014). Macrophage plasticity and polarization in liver homeostasis and pathology. Hepatology 59(5): 2034-2042.
  5. Stijlemans, B., Leng, L., Brys, L., Sparkes, A., Vansintjan, L., Caljon, G., Raes, G., Van Den Abbeele, J., Van Ginderachter, J. A., Beschin, A., Bucala, R. and De Baetselier, P. (2014). MIF contributes to Trypanosoma brucei associated immunopathogenicity development. PLoS Pathog 10(9): e1004414.
  6. Tacke, F. and Zimmermann, H. W. (2014). Macrophage heterogeneity in liver injury and fibrosis. J Hepatol 60(5): 1090-1096.

简介

在内环境稳定中,肝脏对于营养物质(包括糖,脂质,蛋白质和铁)的代谢是关键的,用于清除毒素,并诱导对肠衍生的抗原的免疫耐受。这些功能使肝脏感染由血源性病原体引起的感染,并且导致各种疾病,从毒素和药物诱导的疾病(CC​​l 4,对乙酰氨基酚)到代谢性疾病(脂肪性肝炎,酒精性肝病,胆汁阻塞,胆汁淤积)或自身免疫。慢性肝损伤常常进展为威胁生命的纤维化,并且可以在肝硬化和肝细胞癌中结束(Pellicoro等人,2014)。
肝脏含有构成肝细胞大部分的实质细胞或肝细胞。它还含有非实质结构细胞,例如窦状内皮细胞和大量非实质的先天免疫细胞,主要是单核细胞,嗜中性粒细胞,巨噬细胞,DC,NK和NKT细胞,其在感染的情况下可触发适应性免疫应答或其他致病性侮辱(Jenne和Kubes,2013)。如何调节这种免疫应答决定了急性和慢性肝损伤的程度(Stijlemans等人,2014)。在这种情况下,肝巨噬细胞已经被证明在肝损伤中发挥中心但发散(从启动到分解)功能。(Sica等人,2014)。在过去几年中已经变得清楚的是,肝巨噬细胞由两类,组织驻留巨噬细胞,源自卵黄囊/胎儿肝祖细胞的库普弗细胞(KC)和来源于骨髓衍生的Ly6C的组织浸润巨噬细胞Hi 单核细胞(Jinhoux和Jung,2014; Tacke和Zimmerman,2014)。在肝损伤或修复期间区分KCs的活性与单核细胞衍生的巨噬细胞的活性目前是巨噬细胞领域中的前沿研究课题。事实上,考虑到肝衰竭的临床管理仍然存在问题,预期更好地理解调节肝损伤的免疫机制将允许开发新的治疗方式。在这里,我们描述肝非实质多形核(PMN)和单核骨髓细胞允许其分子和功能表征的隔离技术。

关键字:髓系细胞, Kupffer Cell(KC), 单核细胞源性巨噬细胞, 多形核粒细胞(PMN), 肝损伤

材料和试剂

  1. 7-8周龄雌性C57Black/6小鼠(Janvier Labs)
  2. RPMI-1640培养基(RPMI)(Life Technologies,目录号:52400-041)
  3. 胶原酶III型(Worthington Biochemical,目录号:LS004180)
  4. DNase I(Roche Diagnostics,目录号:04536282001)
  5. 肝素(来自猪肠粘膜的钠盐)(Sigma-Aldrich,目录号H3393-1MU)
  6. 没有钙或镁或酚红的Hank's缓冲盐溶液(HBSS)(Life Technologies,Gibco ,目录号:14175-053)
  7. NH 4 Cl(Merck KGaA,目录号:01145.0500)
  8. KHCO 3(Merck KGaA,目录号:04854.0500)
  9. EDTA(Duchefa Biochemie,目录号:E0511.1000)
  10. HCl(37%储备溶液)(Merck KGaA,目录号:1.00317.1000)
  11. 胎牛血清(FBS)(Biowhittaker /Lonza,目录号:DE14-801F)
  12. Lymphoprep TM (Axis-shield,目录号:1114547)
  13. Percoll TM (GE Healthcare,目录号:17-0891-01)
  14. 纯化的CD16/CD32(Fc区段)(克隆2.4G2)(BD Biosciences,目录号:553142)
  15. PE-Cy7缀合的抗CD11b抗体(克隆M1/70)(BD Biosciences,目录号:552850)
  16. AF647缀合的抗Ly6C抗体(克隆ER-MP20)(Serotec,目录号:MCA2389A647)
  17. PerCP-Cy5.5-结合的抗I-A/I-E(MHC-II)抗体(克隆M5/114.15.2)(Biolegend,目录号:107626)
  18. FITC缀合的抗Ly6G抗体(克隆1A8)(BD Biosciences,目录号:551460)
  19. APC-Cy7缀合的CD45抗体(克隆30-F11)(BD Biosciences,目录号:103116)
  20. PE缀合的F4/80抗体(克隆C1:A3-1)(AbD Serotec,目录号:MCA497PET)
  21. 台盼蓝(BDH Chemicals,目录号:34078)
  22. NaCl(Thermo Fisher Scientific,目录号:10428420)

  23. (Merck KGaA,目录号:1.04873.1000)< />
  24. (Merck KGaA,目录号:1.06580.1000)
    的水溶液中,在室温下, >
  25. L-谷氨酰胺(Sigma-Aldrich,目录号:G8540-100G)
  26. 青霉素(Life Technologies,Gibco ,目录号:15140-122)
  27. 链霉素(Life Technologies,Gibco ,目录号:15140-122)
  28. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  29. 丙酮酸钠(Life Technologies,Gibco ,目录号:11360-039)
  30. 非必需氨基酸(Life Technologies,Gibco ,目录号:11140-035)
  31. 肝消化介质(见配方)
  32. 磷酸盐缓冲盐水(PBS)(见Recipes)
  33. 33%Percoll工作溶液(见配方)
  34. 红细胞裂解缓冲液(参见配方)
  35. MACS缓冲区(参见配方)
  36. 细胞悬浮培养基(参见配方)
  37. 封锁介质(参见配方)
  38. 完整介质(见配方)
  39. 台盼蓝工作溶液(见配方)

设备

  1. 聚酯过滤器切割成10×10cm正方形,螺纹直径70μm(Spectrumlabs,目录号:146490)
  2. 10ml注射器(Omnifix,目录号:473203)
  3. BD Falcon 50ml聚丙烯管(BD Biosciences,目录号:2070)
  4. BD Falcon 15ml聚丙烯管(BD Biosciences,目录号:2096)
  5. BD Falcon 5ml聚丙烯圆底管(BD Biosciences,目录号:352063)
  6. 针(Microlance 22G1,1/2,0.7×40mm)(BD Biosciences,ref:301000)
  7. 无菌培养罩
  8. 外科剪刀和镊子
  9. 37℃,5%CO 2细胞培养箱(Forma Scientific)
  10. 移液器
  11. 离心机(Eppendorf,型号:5810R和5417C)
  12. 轨道摇床(Belgolabo,型号:Julabo型SW-20C)以200rpm使用
  13. 光学显微镜(Olympus,型号:CK2)
  14. 多色流式细胞仪(BD Biosciences,FACSCanto TM
  15. GentleMACS TM解离器(Miltenyi Biotec,目录号:130-093-235)
  16. GentleMACS TM管(Miltenyi Biotec,目录号:130-093-237)

程序

  1. 肝单细胞悬液的制备
    1. 牺牲鼠标使用CO 2并通过将其爪子固定到一个约束   泡沫表面使用注射器针。 值得注意的是,所有鼠类实验 根据ECPVA指南(CETS n°123)进行 批准VUB伦理委员会(许可证编号:08-220-8)。 通过心脏穿刺(〜1ml)采集肝素化的血液以便 防止在服用肝脏时太多的血液污染。 或者,可以通过门静脉在体内灌注肝脏 用10ml盐水。 从尾部的基部做一个平行的切口   到沿着鼠标的腹部和没有爪子的颈部 穿刺腹膜。 轻轻拉回皮肤,并将其固定到 泡沫表面。 随后,打开鼠标腹部,轻轻地移动 肠道在侧面使用棉塞获得肝脏。
    2. 轻轻地从身体取出肝脏,而不损坏它
    3. 将收获的肝脏在冰上的50ml Falcon管中的5ml RPMI培养基中,直到消化过程
    4. 将肝脏置于GentleMACS TM C管中并加入5ml肝消化 中。 随后,使用切成小块(1-1.5mm)的肝脏 (图1A/B)。
    5. 使用肝脏均质化 使用2次程序mLiver_01_03进行GentleMACS TM离解(图1C) (16秒)。随后,在37℃孵育20-30 min,同时在水浴中摇动以允许组织消化
    6. 在室温下使用GentleMACS TM离解剂程序mLiver_02_03(25秒)再次使肝脏悬浮液均质化(图 1D)。最后,加入5ml阻断介质以阻止肝消化。
    7. 为了测量全肝中的细胞因子含量,收集  0.5ml溶液在1.5ml Eppendorf管中并在其离心 10,625×g/g,在室温下8分钟。随后,收集 上清并储存于-20℃直至需要。
    8. 过滤 剩余肝脏悬浮液通过70-μm无菌尼龙纱布进入 无菌50ml锥形管。洗涤GentleMACS TM C管 额外的20ml阻断溶液,并再次冲洗过滤器 有了这个。
    9. 在4℃下以450xg离心50分钟8分钟,轻轻弃去上清液。
    10. 通过在5中重悬细胞沉淀来消除红细胞 ml冰冷的红细胞裂解缓冲液,并在冰上放置2-3分钟。
    11. 通过加入25ml细胞悬浮培养基中和裂解,和 通过70-μm无菌将悬浮液转移到新的50ml管中 尼龙纱布。
    12. 在4℃下以450×g离心50分钟8分钟,轻轻弃去上清液。
    13. 重悬细胞沉淀在5ml细胞悬浮培养基中,并使用台盼蓝计数活细胞。
      在这个阶段,悬浮液含有实质(肝细胞) 和非实质(包括多形核细胞,单核细胞, 巨噬细胞,DC,NK和NKT细胞)肝细胞。 虽然这样 悬浮液可以通过流式细胞术进行分析,建议 进行额外的分级步骤以允许细胞培养和/或 FACS分选。

  2. 使用Lymphoprep梯度分离分离肝细胞
    1. 缓慢加入10 ml Lymphoprep?sup?(Lucron Bioproducts) 使用具有长针的10ml注射器重悬浮肝细胞溶液。 以800×g离心25分钟,在20℃,没有加速或制动 (图1E)。
    2. 小心收集低密度细胞层 包含非实质肝细胞(富含 单核骨髓细胞以及T,B和NK/NKT细胞)。 如果 间期不清晰可见(细胞数量少)你也可以 收集上层相(含RPMI培养基)。 采取少于 可能的下层包含Lymphoprep(图1E)。
    3. 转移中间相到一个新的无菌15毫升管和填充到顶部 与MACS缓冲区。 在20℃下以800×g离心7分钟并弃去 上清液。 重悬细胞沉淀在1-2毫升细胞悬浮液 培养基中,并且在使用台盼蓝计数细胞后, 浓度为10 7个细胞/ml
    4. 去除Lymphoprep后, 收集主要含有实质细胞的低级分(沉淀) (肝细胞)和多形核(PMN)细胞 在非实质细胞部分中由于它们的高密度 特征并将其转移至无菌的15ml Falcon管(图 1E)。将细胞重悬在最终体积为15ml的细胞悬浮液中 并在20℃下以650×g离心8分钟
    5. 随后,为了去除Lymphoprep溶液进行1-2 洗涤步骤为15ml细胞悬浮培养基。最后,重新悬挂 在3ml细胞悬浮培养基中沉淀,并在计数细胞后 使用台盼蓝,使浓度为10 7个细胞/ml。
      的 间期(非实质细胞)部分现在可用于FACS 分析/分选以及细胞培养条件。底端 含有肝细胞和剩余的非实质细胞(PMN  以及具有更高密度的巨噬细胞)也可以用于 流式细胞术分析。后一部分可以进一步净化 使用Percoll梯度以从肝中分离肝细胞 剩余的非实质细胞。值得注意的是,如果不需要肝细胞 DNase 1可以添加到消化培养基中,导致更清洁 样品

  3. 使用Percoll梯度分离分离肝细胞
    1. 要从PMN和剩余的巨噬细胞分离肝细胞,混合1 vol。 (〜5ml的10μL/ml工作溶液)的细胞悬浮液中 vol。 的密度为1.07g/ml的等渗Percoll溶液(33%工作 解)。 在室温下以800×g离心30分钟(图 1F)。 吸出肝细胞,形成宽带的密度 1.07-1.09g/ml,从梯度洗脱除去Percoll两次 在800×g离心5分钟的另外的离心循环 再悬浮于3ml细胞悬浮培养基中
    2. 取出后  Percoll,收集主要含有PMN的下部细胞沉淀部分 和未保留在非实质内的巨噬细胞 由于它们的密度特性并将其转移到a 无菌15ml Falcon管
    3. 随后,在650×g离心5分钟。在4℃下,将沉淀重悬于1ml细胞悬浮培养基中,  使用台盼蓝计数细胞,并使它们的浓度 10 细胞/ml。
      在部分B和C中获得的不同级分 可用于流式细胞术分析或当完全重悬时  培养基用于体外细胞培养

      图1.肝单细胞 制备 A.在5ml消化培养基中的GentleMACS TM管中的肝脏。  B.用剪刀切割后的GentleMACS TM C管中的肝脏。 C。 解离器。 D.肝脏在GentleMACS TM C管中 消化和使用GentleMACS TM离解器均质化。 随后,过滤悬浮液并裂解RBC。 E.渐变之后 离心,具有清晰可见的间期(非实质细胞,   红色箭头)和沉淀(肝细胞和剩余的非实质 细胞具有更高的密度)。 F.可选:Percoll梯度后 用明显可见的上部分(肝细胞,蓝色)离心 箭头)和颗粒(PMN和剩余的非实质细胞具有更高   密度)。

  4. 流式细胞分析
    1. 转移100μl的10 7个细胞/ml不同的储备溶液 级分[(界面Lymphoprep(部分B),沉淀Percoll(部分 C)]加入到5ml聚丙烯圆底管中以防止粘连 细胞。 用1μg大鼠抗小鼠CD16/CD32孵育细胞悬浮液 FcR阻断抗体克隆(2.4G2,每10 6个细胞1μg)在冰水上 20分钟。
    2. 随后,加入荧光标记的抗体 (0.2μg/10 6个细胞)在冰水上再保温20分钟,保护 暴露于光。 使用的抗体是FITC缀合的Ly6G, PE缀合的F4/80,APC缀合的Ly6C,APC-Cy7缀合的CD45, PE-Cy7缀合的抗CD11b和PerCP-Cy5.5缀合的MHC-II
    3. 通过加入2ml冰冷的MACS缓冲液洗涤,在450×g离心6小时 min,并弃去上清液。 加入100-200μl细胞悬浮液 培养基以保持细胞存活,将细胞转移到FACS管中, 继续到FACSCanto TM
    4. 使用FlowJo分析FACS数据 软件。简而言之,在选择生命门和单细胞(使用a  FSC-A对比FSC-H谱)选择CD45中的CD45 + 细胞 FSC-A图(图2,上图和下图)。在CD45 + 细胞内, 基于它们的CD11b pos Ly6G pos 表达谱轮廓选出PMN, 选择CD11b pos Ly6G neg 表达细胞(图3A)。的 CD11b neg Ly6G neg 群主要由T细胞,B细胞和 NK细胞。接下来,在Ly6C相对于MHC-II中绘制CD11b pos Ly6G neg 细胞 并随后检查F4/80表达。因此,单核细胞 Ly6C高/低MHC-II低/低F4/80低细胞,单核细胞衍生的"不成熟"细胞, 巨噬细胞作为Ly6C高/高MHC-II高/高F4/80高/高细胞,常驻/成熟 巨噬细胞(即Kupffer细胞/"成熟的"单核细胞衍生的巨噬细胞)  Ly6C neg/low MHC-II F4/80 高细胞,将单核细胞巡逻 Ly6C neg/low MHC-II neg F4/80 低细胞和嗜酸性粒细胞 鉴定了Ly6C MHC-II neg F4/80 表达(图3B)。的 注意,基于其SiglecF确认嗜酸性粒细胞的身份 表达和高SSC。


      图2.代表性的FACS门控 不同肝制剂的策略(全肝悬液, Lymphoprep上部分,Lymphoprep下部分,Percoll下部分 级分和Percoll上级分)。 (上)选择寿命门 基于SSC-A对FSC-A图。 (下)选择单 细胞,在CD45对FSC图中选择CD45 + 细胞

      图3。 在CD45 +部分内骨髓细胞的代表性门控策略。A.在Ly6G中绘制CD45 sup + +细胞(参见图2,下图)  相对于CD11b图,以鉴定PMN(CD11b pos pos Ly6G pos pos ),单核细胞  (CD11b pos Ly6G neg )和CD11b neg Ly6G neg细胞(T细胞,NK细胞, B细胞)。 B.将剩余的CD11b pos Ly6G neg 级分绘制在 MHC-II对Ly6C图以允许鉴定单核细胞 (Ly6C高 - > MHC-II neg ),单核细胞衍生的"未成熟"巨噬细胞(Ly6C高 - 高MHC-II高 - ),"驻留/成熟"巨噬细胞(库普弗细胞,Ly6C neg/low high ),巡逻单核细胞(Ly6C low/neg MHC-II neg )和嗜酸性粒细胞  (EO,Ly6C MHC-II neg )。 C.炎症单核细胞的F4/80图 (黑色),巡逻单核细胞(蓝色),单核细胞衍生的"不成熟" 巨噬细胞(绿色)和"驻留/成熟"巨噬细胞(橙色)。

食谱

  1. 肝消化介质
    1ml胶原酶III型原液:6,000U/ml)稀释于50ml无钙或镁的Hanks平衡盐溶液(HBSS)中。
    分装胶原酶III型(1ml /管)的储备溶液并在-20℃下冷冻 可选:如果不需要肝细胞,向培养基中加入10单位/ml的DNA酶I。
  2. 磷酸盐缓冲盐水(PBS)
    8g/L NaCl
    0.2 g/L KCl
    0.24g/L KH 2 PO 4 sub/
    1.8g/L Na 2 HPO 4+ 2H 2 O O
    加入蒸馏水至1升,用HCl(储备溶液37%)调节pH值为7.4
  3. 33%Percoll工作溶液
    将PBS加入16.5ml Percoll 储备溶液中,直至最终体积达到终体积为
  4. 红细胞裂解缓冲液
    8.29g/L NH 4 Cl/h 1 g/L KHCO 3
    37.2mg/L EDTA
    加入蒸馏水至1升,用HCl调至pH 7.2
  5. MACS缓冲区
    不含钙或镁或酚红的HBSS 2%(v/v)热灭活的胎牛血清(FBS) 3 mM EDTA
  6. 细胞悬浮培养基
    RPMI
    5%(v/v)热灭活的胎牛血清(FBS)
  7. 封锁媒介
    不含钙或镁或酚红的HBSS 2%(v/v)热灭活的FBS 5 mM EDTA
  8. 完成媒介
    罗斯威尔公园纪念研究所(RPMI)-1640
    10%(v/v)热灭活的胎牛血清(FBS) 300μg/ml L-谷氨酰胺 100 U/ml青霉素
    100μg/ml链霉素 0.02mMβ-巯基乙醇 1mM丙酮酸钠 1 mM非必需氨基酸
  9. 台盼蓝工作溶液
    在PBS中制备1%v/v台盼蓝溶液,并以1/10比例使用(细胞/台盼蓝)

致谢

我们承认国际大学吸引人计划(PAI-IAP N. P7/41, http://www.belspo.be/belspo/iap/index_en.stm )和来自FWO的授权(KaN 1511812N)。 Benoit Stijlemans是由VUB/SRP针对与传染病和癌症相关的炎症(纳米抗体健康)支持的研究员。 作者还感谢Ella Omasta,Marie-Therese Detobel,Maria Slazak,Victor Orimoloye和Nadia Abou的技术援助。 我们还要感谢Carl De Trez博士的建设性讨论。

参考文献

  1. Ginhoux,F.和Jung,S。(2014)。 单核细胞和巨噬细胞:发育途径和组织内环境稳定。 Nat Rev Immunol 14(6):392-404。
  2. Jenne,C.N.和Kubes,P。(2013)。 肝脏的免疫监视。 14 Nat Immunol 14 10):996-1006。
  3. Pellicoro,A.,Ramachandran,P.,Iredale,J.P。和Fallowfield,J.A。(2014)。 肝纤维化和修复:实体器官中伤口愈合的免疫调节。 Nat Rev Immunol 14(3):181-194。
  4. Sica,A.,Invernizzi,P。和Mantovani,A。(2014)。 巨噬细胞在肝脏动态平衡和病理学中的可塑性和极化。 肝病学 59(5):2034-2042。
  5. Stijlemans,B.,Leng,L.,Brys,L.,Sparkes,A.,Vansintjan,L.,Caljon,G.,Raes,G.,Van Den Abbeele,J.,Van Ginderachter,JA,Beschin,A 。,Bucala,R。和De Baetselier,P。(2014)。 MIF有助于
    相关的免疫致病性发展。 em> PLoS Pathog 10(9):e1004414。
  6. Tacke,F。和Zimmermann,H.W。(2014)。 肝损伤和纤维化中的巨噬细胞异质性。 Hepatol 60(5):1090-1096
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Stijlemans, B., Sparkes, A., Abels, C., Keirsse, J., Brys, L., Elkrim, Y., Baetselier, P. D., Beschin, A. and Ginderachter, J. A. (2015). Murine Liver Myeloid Cell Isolation Protocol . Bio-protocol 5(10): e1471. DOI: 10.21769/BioProtoc.1471.
  2. Stijlemans, B., Leng, L., Brys, L., Sparkes, A., Vansintjan, L., Caljon, G., Raes, G., Van Den Abbeele, J., Van Ginderachter, J. A., Beschin, A., Bucala, R. and De Baetselier, P. (2014). MIF contributes to Trypanosoma brucei associated immunopathogenicity development. PLoS Pathog 10(9): e1004414.
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