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Preparation of Single Cell Suspensions from Mouse Aorta
从小鼠主动脉制备单细胞悬浮液   

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Abstract

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner et al., 2009; Hu et al., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewa et al., 2016; Galkina et al., 2006; Lotzer et al., 2010; Mohanta et al., 2016; Mohanta et al., 2014; Moos et al., 2005; Srikakulapu et al., 2016; Zhao et al., 2004). There are conflicting reports on the number and subtypes of immune cells in the aorta depending on the age of the animals, the protocol that is used to obtain single cell suspensions, and the dietary conditions of the mice (Campbell et al., 2012; Clement et al., 2015; Galkina et al., 2006; Kyaw et al., 2012). The number of immune cells in the aorta differs as much as tenfold using different protocols (Butcher et al., 2012; Galkina et al., 2006; Gjurich et al., 2015; Grabner et al., 2009; Hu et al., 2015). These discrepant results call for a protocol that robustly documents bona fide aorta cells rather than those in the surrounding tissues or blood. Critical methodological hurdles include the removal of adjacent adipose tissue and small paraaortic lymph nodes lining the entire aortic tree that are not visible by the naked eye. A dissection microscope is therefore recommended. Moreover protocols of aorta preparations should ascertain that lymphocyte aggregates referred to as fat associated lymphoid clusters (FALCs) (Benezech et al., 2015; Elewa et al., 2015) that are often present at the border between the adipose tissue and the adventitia are removed before enzyme digestion. We propose - besides other approaches (Hu et al., 2015; Mohanta et al., 2014) - a combination of immunohistochemical staining and fluorescence activated cell sorter (FACS) analyses from single cell suspensions to quantify the cells of interest. This protocol describes isolation of single cells from mouse aorta for FACS and other analysis.

Materials and Reagents

  1. 50 ml Falcon tube (VWR International, CellStar®, catalog number: 188271 )
  2. 100 µm cell strainer (BD, catalog number: 352360 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 352360”.
  3. 1 ml syringe (Henke-Sass, Wolf GmbH, Soft-JECT®, catalog number: 5010-200V0 )
  4. 5 ml syringe (BD, catalog number: 309646 )
  5. Needle-26G (B. Braun Medical Inc., catalog number: 4657683 )
  6. 6-well plate (BD Falcon, catalog number: 353046 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353046”.
  7. 1.5 ml Eppendorf tube (Eppendorf AG, catalog number: 0030123328 )
  8. Trypan blue solution (Sigma-Aldrich, catalog number: 93595 )
  9. Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  10. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040133 )
  11. Fetal bovine serum (FBS) (PAN Biotech UK Ltd., catalog number: P30-1506 )
  12. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  13. Collagenase from Clostridium histolyticum, type I (Sigma-Aldrich, catalog number: C0130 )
  14. Collagenase from Clostridium histolyticum, type XI (Sigma-Aldrich, catalog number: C7657 )
  15. Hyaluronidase from bovine testes, type I-s (Sigma-Aldrich, catalog number: H3506 )
  16. DNase I (Sigma-Aldrich, catalog number: 11284932001 )
  17. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630106 )
  18. Ethanol solution (Sigma-Aldrich, catalog number: 48075 )
  19. Anti-mouse CD45 APC antibody (Thermo Fisher Scientific, eBioscience, catalog number: 17-0451-82 )
  20. LIVE/DEAD® fixable blue dead cell stain kit (Invitrogen, catalog number: L23105 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L23105”.
  21. Fc block (anti-CD16/32) (Thermo Fisher Scientific, eBioscience, catalog number: 16-0161-82 )
  22. FACS buffer (see Recipes)
  23. EDTA buffer (see Recipes)
  24. Enzyme cocktail (see Recipes)

Equipment

  1. Dissecting scissors (Fine Science Tools, catalog number: 91460-11 )
  2. Curved forceps (Fine Science Tools, catalog number: 11073-10 )
  3. CO2 supply machine (Next Advance, model: Quietek CO2 induction system )
  4. Neubauer cell counting chamber (Marienfeld-Superior)
  5. Microscope (Carl Zeiss Microscopy, model: Axiovert 40C )
  6. Dissecting microscope equipped with cold light (Carl Zeiss Microscopy, model: Stemi2000 )
  7. Water bath (Thomas Scientific, model: 1196x11 )
  8. BD LSRFortessa (BD Bioscience)

Procedure

  1. Isolation of mouse aorta
    1. Euthanize mouse by CO2 as approved by the appropriate Animal Care and Use Committee.
    2. Place the mouse in supine position and fix arms and legs onto foam plate with needles.
    3. Spray 75% ethanol onto abdominal fur, and draw blood by cardiac puncture using 26G needle fixed to a 1 ml syringe.
    4. Cut skin, subcutaneous tissue, and open peritoneal cavity from abdomen to thorax along the middle line; fix skin/subcutaneous tissue onto a foam plate with needles.
    5. Open the mediastinum and cut off ribs.
    6. Puncture the right atrium using a 26G needle.
    7. Use a 5 ml syringe with a 26G needle attached, perfuse the remaining blood in the aorta from left ventricle with 5 ml 2 mM EDTA buffer, 10 ml PBS, and 10 ml FACS buffer, respectively. To avoid blood contamination, additional 3 perfusions using 5 ml FACS buffer each are needed during dissection steps. Following this, the aorta should be white.
    8. Remove intestine, spleen, liver, lung, and leave heart, kidneys, and aorta in situ.
    9. Under a dissecting microscope equipped with a cold light source (magnification, 2.5x), carefully dissect and remove adipose tissue that is located adjacent to the adventitia while leaving the aorta adventitia intact. At this step, there is no morphologically definable tissue identifier to distinguish adventitia and adjacent adipose tissue (Figure 1). However the adipose tissue is soft and can be removed easily; aorta adventitia is tight.


      Figure 1. Aorta anatomy in situ. Aorta was dissected from a 78-week old Apoe-/- mouse. Artery branches are indicated. Blue dashed line indicates position of diaphragm. The plaques in aorta segments are white indicated by asterisks. The adjacent adipose tissue is indicated by blue arrows.

    10. Carefully remove any lymph nodes that are close to the aorta (Figure 2). Lymph nodes are light brown. There are also some small lymph nodes in adjacent adipose tissue that are not visible with the naked eye requiring a dissection microscope for detection and removal.


      Figure 2. Lymph nodes adjacent to aorta. Two lumbar lymph nodes adjacent to the abdominal aorta adventitia are indicated by black arrows. There are also invisible small lymph nodes close to or within the paraaortic adipose tissue that line the entire paraaortic connective tissue.

    11. Main artery branches should be left untouched (0.5 cm long from their branching location): Innominate artery, left carotid artery, left subclavian artery, renal arteries, celiac artery, and the common iliac artery (Figure 1). 
    12. Harvest whole aorta and cut it at the level of diaphragm (Figure 1) into two segments: Abdominal aorta and thoracic aorta if analyses of individual aorta segments is needed.
    13. To study cells from the adventitia vs plaques, detachment of plaque (Figure 1) from the intima is needed: Open aorta longitudinally, fix it with needles, and place the intima side en face; carefully remove the plaque with a curved forceps. Plaque is white, easy to distinguish, and removed without effort.
    14. Keep aorta segments in ice-cold FACS buffer in 6-well plates until enzyme digestion.

  2. Enzyme digestion (Grabner et al., 2009; Hu et al., 2015)
    1. Enzyme cocktail: 400 U/ml collagenase type I, 120 U/ml collagenase type XI, 60 U/ml hyaluronidase and 60 U/ml DNase1, 20 mM HEPES in Dulbecco’s phosphate buffered saline (DPBS) containing calcium.
    2. Transfer aorta from the 6-well plate to a 1.5 ml Eppendorf tube containing 0.5 ml enzyme cocktail. Cut aorta tissue into small pieces using scissors.
    3. Transfer enzyme cocktail with aorta tissue to a 50 ml Falcon tube. Add additional 2 ml enzyme cocktail (2.5 ml enzyme cocktail for each thoracic and abdominal aorta total).
    4. Transfer tube containing aorta tissue pieces to a water bath for 50 min digestion at 37 °C with slow shaking.

  3. Prepare immune cell suspension from aorta
    1. After 50 min, pour the digestion solution onto a 100 µm cell strainer which is placed on the top of a new 50 ml Falcon tube.
    2. Mash remaining aorta tissue with syringe plunger and rinse cell strainer with 5 ml FACS buffer.
    3. Collect flowthrough from steps C1-2 and centrifuge at 300 x g, 4 °C for 10 min.
    4. Carefully remove supernatant after centrifugation, and resuspend cell pellet in 2 ml FACS buffer.
    5. Count aortic cells after mixing with trypan blue under a light microscope.

  4. Aorta cell staining and flow cytometry
    1. Centrifuge cell suspension at 300 x g, 4 °C for 5 min.
    2. To block Fc receptors, incubate cell pellet in 100 µl 1:100 diluted anti-CD16/32 mAb in FACS buffer (1 µg/ml final concentration) at 4 °C for 20 min.
    3. Fill the staining tube with 300 µl FACS buffer and centrifuge as step D1.
    4. Discard the supernatant and incubate the cell pellet with 100 µl 1:200 diluted anti-mouse CD45 mAb in FACS buffer (1 µg/ml final concentration) at 4 °C for 20 min.
    5. Fill the staining tube with 300 µl FACS buffer and centrifuge as in step D1.
    6. Resuspend the cell pellet with 200 µl FACS buffer and keep the sample on ice until FACS.
    7. To exclude dead cells, DAPI, PI or LIVE/DEAD cell viability assay dye, like Indo-1, is needed to stain with the cells before measurement.

Notes

  1. To avoid cell contamination from blood or tissues adjacent to the aorta, the perfusion steps and removal of associated adipose tissue as well as lymph nodes should be done very carefully under the microscope. IF staining showed that FALCs are present in the paraaortic adipose tissue sometimes close to the adventitia.
  2. Blood should be completely flushed out of the aorta before digestion. EDTA buffer prevents blood clotting, and it is initially used for perfusing the aorta. However, EDTA can inhibit the activity of digestion enzymes that require Ca++; EDTA needs to be flushed out before digestion is initiated.
  3. During dissection of the aorta, intermittently spray FACS buffer onto the aorta to prevent drying.
  4. Calcium is required for collagenase activity. Therefore, DPBS with calcium is recommended for preparing the enzyme cocktail.
  5. The total number of aortic cells after digestion varies depending on age, sex, diet, and mouse genotype.
  6. Enzyme activity may differ from batch to batch. Enzyme activity tests of each batch are needed.
  7. Enzyme digestion can remove some surface antigens. Therefore, it is necessary to test each surface marker after enzyme digestion. For example, CD138 detection will be low after digestion.
  8. During digestion, some cells will undergo apoptosis. Therefore, flow cytometry staining of live/dead cells is strongly recommended using DAPI, PI or other dye.
  9. After digestion, tissue fragments will be generated. In order to get ‘clean’ FACS plots, anti-mouse CD45 mAb (a panleukocyte marker) and Indo-1 (a marker for dead cell staining) are added and gated (Figure 3).


    Figure 3. FACS profile and gating for living CD45+ cells. Aorta single cell suspensions were prepared from a 78 week-old Apoe-/- mouse. Cells were stained with anti-CD45 mAb and dead cells using Indo-1.

Recipes

  1. FACS buffer
    PBS + 2% FBS
  2. EDTA buffer
    PBS + 2 mM EDTA
  3. Enzyme cocktail
    400 U/ml collagenase type I
    120 U/ml collagenase type XI
    60 U/ml hyaluronidase and 60 U/ml DNase1
    20 mM HEPES in Dulbecco’s phosphate buffered saline (DPBS) containing calcium

Acknowledgments

This work was supported by the German Research Council (HA 1083/15-4 to A.J.R.H.; MO 3052/1-1 to S.M., and YI 133/2-1 to C.Y.) and the European Research Council (AdG 249929 to C.W.).

References

  1. Benezech, C., Luu, N. T., Walker, J. A., Kruglov, A. A., Loo, Y., Nakamura, K., Zhang, Y., Nayar, S., Jones, L. H., Flores-Langarica, A., McIntosh, A., Marshall, J., Barone, F., Besra, G., Miles, K., Allen, J. E., Gray, M., Kollias, G., Cunningham, A. F., Withers, D. R., Toellner, K. M., Jones, N. D., Veldhoen, M., Nedospasov, S. A., McKenzie, A. N. and Caamano, J. H. (2015). Inflammation-induced formation of fat-associated lymphoid clusters. Nat Immunol 16(8): 819-828.
  2. Butcher, M. J., Gjurich, B. N., Phillips, T. and Galkina, E. V. (2012). The IL-17A/IL-17RA axis plays a proatherogenic role via the regulation of aortic myeloid cell recruitment. Circ Res 110(5): 675-687.
  3. Campbell, K. A., Lipinski, M. J., Doran, A. C., Skaflen, M. D., Fuster, V. and McNamara, C. A. (2012). Lymphocytes and the adventitial immune response in atherosclerosis. Circ Res 110(6): 889-900.
  4. Clement, M., Guedj, K., Andreata, F., Morvan, M., Bey, L., Khallou-Laschet, J., Gaston, A. T., Delbosc, S., Alsac, J. M., Bruneval, P., Deschildre, C., Le Borgne, M., Castier, Y., Kim, H. J., Cantor, H., Michel, J. B., Caligiuri, G. and Nicoletti, A. (2015). Control of the T follicular helper-germinal center B-cell axis by CD8(+) regulatory T cells limits atherosclerosis and tertiary lymphoid organ development. Circulation 131(6): 560-570.
  5. Elewa, Y. H., Ichii, O. and Kon, Y. (2016). Comparative analysis of mediastinal fat-associated lymphoid cluster development and lung cellular infiltration in murine autoimmune disease models and the corresponding normal control strains. Immunology 147(1): 30-40.
  6. Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I. J. and Ley, K. (2006). Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med 203(5): 1273-1282.
  7. Gjurich, B. N., Taghavie-Moghadam, P. L. and Galkina, E. V. (2015). Flow cytometric analysis of immune cells within murine aorta. Methods Mol Biol 1339: 161-175.
  8. Grabner, R., Lotzer, K., Dopping, S., Hildner, M., Radke, D., Beer, M., Spanbroek, R., Lippert, B., Reardon, C. A., Getz, G. S., Fu, Y. X., Hehlgans, T., Mebius, R. E., van der Wall, M., Kruspe, D., Englert, C., Lovas, A., Hu, D., Randolph, G. J., Weih, F. and Habenicht, A. J. (2009). Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice. J Exp Med 206(1): 233-248.
  9. Hu, D., Mohanta, S. K., Yin, C., Peng, L., Ma, Z., Srikakulapu, P., Grassia, G., MacRitchie, N., Dever, G., Gordon, P., Burton, F. L., Ialenti, A., Sabir, S. R., McInnes, I. B., Brewer, J. M., Garside, P., Weber, C., Lehmann, T., Teupser, D., Habenicht, L., Beer, M., Grabner, R., Maffia, P., Weih, F. and Habenicht, A. J. (2015). Artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell lymphotoxin beta receptors. Immunity 42(6): 1100-1115.
  10. Kyaw, T., Tay, C., Hosseini, H., Kanellakis, P., Gadowski, T., MacKay, F., Tipping, P., Bobik, A. and Toh, B. H. (2012). Depletion of B2 but not B1a B cells in BAFF receptor-deficient ApoE mice attenuates atherosclerosis by potently ameliorating arterial inflammation. PLoS One 7(1): e29371.
  11. Lotzer, K., Dopping, S., Connert, S., Grabner, R., Spanbroek, R., Lemser, B., Beer, M., Hildner, M., Hehlgans, T., van der Wall, M., Mebius, R. E., Lovas, A., Randolph, G. J., Weih, F. and Habenicht, A. J. (2010). Mouse aorta smooth muscle cells differentiate into lymphoid tissue organizer-like cells on combined tumor necrosis factor receptor-1/lymphotoxin beta-receptor NF-kappaB signaling. Arterioscler Thromb Vasc Biol 30(3): 395-402.
  12. Mohanta, S., Yin, C., Weber, C., Hu, D. and Habenicht, A. J. R. (2016). Aorta atherosclerosis lesion analysis in hyperlipidemic mice. Bio-protocol 6(11): e1833.
  13. Mohanta, S. K., Yin, C., Peng, L., Srikakulapu, P., Bontha, V., Hu, D., Weih, F., Weber, C., Gerdes, N. and Habenicht, A. J. (2014). Artery tertiary lymphoid organs contribute to innate and adaptive immune responses in advanced mouse atherosclerosis. Circ Res 114(11): 1772-1787.
  14. Moos, M. P., John, N., Grabner, R., Nossmann, S., Gunther, B., Vollandt, R., Funk, C. D., Kaiser, B. and Habenicht, A. J. (2005). The lamina adventitia is the major site of immune cell accumulation in standard chow-fed apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 25(11): 2386-2391.
  15. Srikakulapu, P., Hu, D., Yin, C., Mohanta, S. K., Vineela Bontha, S., Peng, L., Beer, M., Weber, C., McNamara, C. A., Grassia, G., Maffia, P., Manz, R. A. and Habenicht, A. J. (2016). Artery tertiary lymphoid organs control multilayered territorialized atherosclerosis B-Cell responses in aged ApoE ITALIC! -/- mice. Arterioscler Thromb Vasc Biol.
  16. Zhao, L., Moos, M. P., Grabner, R., Pedrono, F., Fan, J., Kaiser, B., John, N., Schmidt, S., Spanbroek, R., Lotzer, K., Huang, L., Cui, J., Rader, D. J., Evans, J. F., Habenicht, A. J. and Funk, C. D. (2004). The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med 10(9): 966-973.

简介

动脉粥样硬化是动脉壁的慢性炎性疾病,其特征在于脂质沉积,斑块形成和免疫细胞浸润。先天性和适应性免疫细胞在疾病发展期间浸润动脉。此外,晚期疾病导致外膜中动脉三级淋巴器官的形成(Grabner等人,2009; Hu等人,2015)。已经在主动脉外膜vs动脉粥样硬化斑块中鉴定了各种不同类型的免疫细胞(Elewa等人,2016; Galkina等人,2006; Lotzer等人, 2010; Mohanta等人,2016; Mohanta等人,2014; Moos等人,2010; Mohanta等人,2010; 2005; Srikakulapu et al。,2016; Zhao et al。,2004)。根据动物的年龄,用于获得单细胞悬浮液的方案和小鼠的饮食条件,存在关于主动脉中免疫细胞的数量和亚型的矛盾报告(Campbell等, 2012; Clement等人,2015; Galkina等人,2006; Kyaw等人,2012)。使用不同的方案,主动脉中免疫细胞的数目差异多达十倍(Butcher等,2012; Galkina等,2006; Gjurich等,2015; Grabner等人,2009; Hu等人,2015)。这些不同的结果要求一个强大地记录真正的主动脉细胞而不是周围组织或血液中的细胞的方案。关键的方法障碍包括去除邻近的脂肪组织和整个主动脉树上的肉眼不可见的小的主动脉旁淋巴结。因此建议使用解剖显微镜。此外,主动脉制备物的方案应确定称为与脂肪相关的淋巴细胞簇(FALC)的淋巴细胞聚集体(Benezech等人,2015; Elewa等人, 2015),其在脂肪组织和外膜之间的边界处经常存在,在酶消化之前被除去。除了其他方法(Hu等人,2015; Mohanta等人,2014) - 我们建议 - 免疫组织化学染色和荧光激活细胞分选(FACS)分析的组合从单细胞悬浮液中定量感兴趣的细胞。该协议描述了从小鼠主动脉分离单个细胞用于FACS和其他分析。

材料和试剂

  1. 50ml Falcon管(VWR International,CellStar ,目录号:188271)
  2. 100μm细胞过滤器(BD,目录号:352360)
    注意:目前,"Corning,Falcon ® ,目录号:352360"
  3. 1ml注射器(Henke-Sass,Wolf GmbH,Soft-JECT ,目录号:5010-200V0)
  4. 5ml注射器(BD,目录号:309646)
  5. Needle-26G(B.Braun Medical Inc.,目录号:4657683)
  6. 6孔板(BD Falcon,目录号:353046) 注意:目前,"Corning,Falcon ® ,目录号:353046"。
  7. 1.5ml Eppendorf管(Eppendorf AG,目录号:0030123328)
  8. 台盼蓝溶液(Sigma-Aldrich,目录号:93595)
  9. 磷酸盐缓冲盐水(PBS),pH 7.4(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  10. Dulbecco's磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM ,目录号:14040133)
  11. 胎牛血清(FBS)(PAN Biotech UK Ltd.,目录号:P30-1506)
  12. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  13. 来自溶组织梭菌(Clostridium histolyticum)I型(Sigma-Aldrich,目录号:C0130)的胶原酶
  14. 来自溶组织梭菌(Clostridium histolyticum)III型(Sigma-Aldrich,目录号:C7657)的胶原酶
  15. 来自牛睾丸的透明质酸酶,I型(Sigma-Aldrich,目录号:H3506)
  16. Dnase I(Sigma-Aldrich,目录号:11284932001)
  17. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(1μM)(Thermo Fisher Scientific,Gibco TM,目录号:15630106)
  18. 乙醇溶液(Sigma-Aldrich,目录号:48075)
  19. 抗小鼠CD45 APC抗体(Thermo Fisher Scientific,eBioscience,目录号:17-0451-82)
  20. LIVE/DEAD 可固定蓝色死细胞染色试剂盒(Invitrogen,目录号:L23105)
    注意:目前,它是"Thermo Fisher Scientific,Molecular Probes TM ,目录号:L23105"。
  21. Fc嵌段(抗CD16/32)(Thermo Fisher Scientific,eBioscience,目录号:16-0161-82)
  22. FACS缓冲区(参见配方)
  23. EDTA缓冲液(见配方)
  24. 酶鸡尾酒(见食谱)

设备

  1. 解剖剪刀(Fine Science Tools,目录号:91460-11)
  2. 弯曲钳(Fine Science Tools,目录号:11073-10)
  3. CO 2供应机器(下一个前进,型号:Quietek CO 2感应系统)
  4. Neubauer细胞计数室(Marienfeld-Superior)
  5. 显微镜(Carl Zeiss显微镜,型号:Axiovert 40C)
  6. 装备有冷光(Carl Zeiss显微镜,型号:Stemi2000)的解剖显微镜
  7. 水浴(Thomas Scientific,型号:1196x11)
  8. BD LSRFortessa(BD Bioscience)

程序

  1. 小鼠主动脉的隔离
    1. 由适当的动物护理和使用委员会批准的CO 2安乐死小鼠
    2. 将鼠标放置在仰卧位置,用针固定手臂和腿到泡沫板上。
    3. 喷洒75%乙醇到腹部皮肤,并通过心脏穿刺抽取血液使用固定到1毫升注射器的26G针。
    4. 沿中线切开皮肤,皮下组织,从腹部到胸部开腹腹腔; 将皮肤/皮下组织固定到带针的泡沫板上
    5. 打开纵隔,切断肋骨。
    6. 使用26G针穿刺右心房。
    7. 使用连接有26G针的5ml注射器,分别用5ml 2mM EDTA缓冲液,10ml PBS和10ml FACS缓冲液灌注来自左心室的主动脉中的剩余血液。为了避免血液污染,在解剖步骤期间需要使用5ml FACS缓冲液的额外3次灌注。接下来,主动脉应该是白色的。
    8. 清除肠,脾,肝,肺,原位离开心脏,肾脏和主动脉。。
    9. 在配备有冷光源(放大倍数,2.5x)的解剖显微镜下,仔细解剖和去除位于外膜附近的脂肪组织,同时保持主动脉外膜完整。在这一步,没有形态学上可定义的组织标识符来区分外膜和相邻的脂肪组织(图1)。然而,脂肪组织是软的并且可以容易地去除;主动脉外膜狭窄。


      图1.原位解剖 。从78周龄的Apoe -/- 小鼠解剖主动脉。指示动脉分支。蓝色虚线表示隔膜的位置。主动脉段中的斑块是白色,用星号表示。相邻的脂肪组织用蓝色箭头表示
    10. 小心地去除靠近主动脉的任何淋巴结(图2)。淋巴结为浅棕色。在相邻脂肪组织中还存在一些小的淋巴结,用肉眼不可见,需要解剖显微镜用于检测和去除。


      图2.与主动脉相邻的淋巴结。邻近腹主动脉外膜的两个腰淋巴结由黑色箭头指示。在主动脉脂肪组织内或靠近整个主动脉旁结缔组织的也有不可见的小淋巴结。

    11. 主动脉分支应保持不变(距离其分支位置0.5厘米长):无效动脉,左颈动脉,左锁骨下动脉,肾动脉,腹腔动脉和髂总动脉(图1)。
    12. 收获整个主动脉,并将其在隔膜水平(图1)切成两个部分:腹主动脉和胸主动脉,如果需要分析个别主动脉段。
    13. 为了研究外膜与斑块的细胞,需要从内膜中分离斑块(图1):纵向打开主动脉,用针固定,并将内膜侧面置于 小心地用弯曲的镊子去除斑块。斑块是白色的,容易区分,并且毫不费力地去除
    14. 保持主动脉段在冰冷的FACS缓冲液在6孔板,直到酶消化
  2. 酶消化(Grabner等人,2009; Hu等人,2015)
    1. 酶混合物:400U/ml I型胶原酶,120U/ml XI型胶原酶,60U/ml透明质酸酶和60U/ml DNase1,20mM HEPES的含有钙的Dulbecco磷酸盐缓冲盐水(DPBS)中。
    2. 将主动脉从6孔板转移到含有0.5ml酶混合物的1.5ml Eppendorf管中。 使用剪刀将主动脉组织切成小块
    3. 将主动脉组织的酶混合物转移到50ml Falcon管中。 加入另外的2ml酶混合物(2.5ml酶混合物用于每个胸部和腹主动脉总)。
    4. 将含有主动脉组织碎片的管子在37℃缓慢振荡下转移到水浴中消化50分钟
  3. 从主动脉制备免疫细胞悬浮液
    1. 50分钟后,将消化溶液倒入一个100微米的细胞过滤器,放置在一个新的50毫升Falcon管的顶部。
    2. 用注射器柱塞和冲洗细胞过滤器用5ml FACS缓冲液捣碎保留的主动脉组织。
    3. 从步骤C1-2收集流出物并在300×g离心,4℃10分钟。
    4. 离心后小心地除去上清液,并将细胞沉淀重悬于2ml FACS缓冲液中
    5. 在光学显微镜下与台盼蓝混合后计数主动脉细胞
  4. 主动脉细胞染色和流式细胞术
    1. 在300×g离心细胞悬浮液,4℃5分钟
    2. 为了阻断Fc受体,将细胞沉淀在100μl1:100稀释的抗CD16/32mAb的FACS缓冲液(1μg/ml终浓度)中在4℃下孵育20分钟。
    3. 用300μlFACS缓冲液填充染色管,离心作为步骤D1
    4. 弃去上清液和细胞沉淀与100微升1:200稀释的抗小鼠CD45单克隆抗体在FACS缓冲液(1微克/毫升终浓度)在4℃孵育20分钟。
    5. 用300μlFACS缓冲液填充染色管,并如步骤D1中离心
    6. 用200μlFACS缓冲液重悬细胞沉淀,将样品保存在冰上直到FACS
    7. 为了排除死细胞,需要在测量前用细胞染色DAPI,PI或LIVE/DEAD细胞活力测定染料,如Indo-1。

笔记

  1. 为了避免来自邻近主动脉的血液或组织的细胞污染,应当在显微镜下非常小心地进行灌注步骤和相关脂肪组织以及淋巴结的去除。 IF染色显示FALC存在于有时接近外膜的主动脉脂肪组织中
  2. 血液应该在消化前完全从主动脉冲出。 EDTA缓冲液防止血液凝固,并且其最初用于灌注主动脉。然而,EDTA可抑制需要Ca ++ 的消化酶的活性;在消化开始前,需要冲洗出EDTA。
  3. 在主动脉解剖期间,间歇地喷雾FACS缓冲液到主动脉,以防止干燥
  4. 钙是胶原酶活性所需的。因此,建议使用含有钙的DPBS来制备酶混合物
  5. 消化后的主动脉细胞总数取决于年龄,性别,饮食和小鼠基因型
  6. 不同批次的酶活性可能不同。需要每个批次的酶活性测试
  7. 酶消化可以去除一些表面抗原。因此,必须在酶消化后测试每个表面标志物。例如,消化后CD138检测将低。
  8. 在消化期间,一些细胞将经历凋亡。因此,强烈建议使用DAPI,PI或其他染料对活细胞/死细胞进行流式细胞术染色。
  9. 消化后,将产生组织碎片。为了获得"清洁的"FACS图,加入抗小鼠CD45mAb(白细胞标志物)和Indo-1(死细胞染色的标志物)并门控(图3)。


    图3.活的CD45 +细胞的FACS图谱和门控。从78周龄的Apoe -/- 小鼠制备主动脉单细胞悬浮液,老鼠。使用Indo-1用抗CD45mAb和死细胞染色细胞。

食谱

  1. FACS缓冲区
    PBS + 2%FBS
  2. EDTA缓冲液
    PBS + 2mM EDTA
  3. 酶混合物
    400U/ml I型胶原酶
    120 U/ml XI型胶原酶 60U/ml透明质酸酶和60U/ml DNA酶1 20mM HEPES在含有钙的Dulbecco's磷酸盐缓冲盐水(DPBS)中

致谢

这项工作由德国研究委员会(HA 1083/15-4至A.J.R.H; MO 3052/1-1至S.M.和YI 133/2-1至C.Y.)和欧洲研究委员会(AdG 249929至C.W.)支持。

参考文献

  1. Benezech,C.,Luu,NT,Walker,JA,Kruglov,AA,Loo,Y.,Nakamura,K.,Zhang,Y.,Nayar,S.,Jones,LH,Flores-Langarica,A.,McIntosh, A.,Marshall,J.,Barone,F.,Besra,G.,Miles,K.,Allen,JE,Gray,M.,Kollias,G.,Cunningham,AF,Withers,DR,Toellner,KM,Jones ,ND,Veldhoen,M.,Nedospasov,SA,McKenzie,AN and Caamano,JH(2015)。  炎症诱导的脂肪相关淋巴细胞簇的形成。自身免疫 16(8):819-828。
  2. Butcher,M.J.,Gjurich,B.N.,Phillips,T.and Galkina,E.V。(2012)。 IL-17A/IL-17RA轴具有促动脉粥样硬化的作用主动脉髓样细胞募集的调节。 Circ Res 110(5):675-687。
  3. Campbell,K.A.,Lipinski,M.J.,Doran,A.C.,Skaflen,M.D.,Fuster,V。和McNamara,C.A。(2012)。 淋巴细胞和动脉粥样硬化中的外膜免疫应答。 Circ Res 110(6):889-900。
  4. Clements,M.,Guedj,K.,Andreata,F.,Morvan,M.,Bey,L.,Khallou-Laschet,J.,Gaston,AT,Delbosc,S.,Alsac,JM,Bruneval, Deschildre,C.,Le Borgne,M.,Castier,Y.,Kim,HJ,Cantor,H.,Michel,JB,Caligiuri,G.and Nicoletti, 通过以下方式控制T滤泡辅助生物中心B细胞轴CD8(+)调节性T细胞限制动脉粥样硬化和三级淋巴器官发育。 循环 131(6):560-570。
  5. Elewa,Y.H.,Ichii,O.and Kon,Y。(2016)。 纵隔脂肪相关淋巴细胞簇发育和肺细胞浸润的比较分析在鼠自身免疫疾病模型和相应的正常对照菌株中。 Immunology 147(1):30-40。
  6. Galkina,E.,Kadl,A.,Sanders,J.,Varughese,D.,Sarembock,I.J.and Ley,K。(2006)。 在动脉粥样硬化发展之前和期间淋巴细胞募集到主动脉壁中是部分L-选择素依赖性。 J Exp Med 203(5):1273-1282。
  7. Gjurich,B.N.,Taghavie-Moghadam,P.L.and Galkina,E.V。(2015)。 小鼠主动脉内免疫细胞的流式细胞术分析。 Methods Mol Biol 1339:161-175。
  8. Grabner,R.,Lotzer,K.,Dopping,S.,Hildner,M.,Radke,D.,Beer,M.,Spanbroek,R.,Lippert,B.,Reardon,CA,Getz,GS, YX,Hehlgans,T.,Mebius,RE,van der Wall,M.,Kruspe,D.,Englert,C.,Lovas,A.,Hu,D.,Randolph,GJ,Weih,F。和Habenicht,AJ (2009)。 淋巴毒素β受体信号传导促进老年主动脉外膜中的三级淋巴器官发生ApoE -/- 小鼠。 J Exp Med 206(1):233-248。
  9. Hu,D.,Mohanta,SK,Yin,C.,Peng,L.,Ma,Z.,Srikakulapu,P.,Grassia,G.,MacRitchie,N.,Dever,G.,Gordon,P.,Burton ,BL,Ialenti,A.,Sabir,SR,McInnes,IB,Brewer,JM,Garside,P.,Weber,C.,Lehmann,T.,Teupser,D.,Habenicht, Grabner,R.,Maffia,P.,Weih,F。和Habenicht,AJ(2015)。 动脉三级淋巴器官控制主动脉免疫并通过血管平滑保护免受动脉粥样硬化肌细胞淋巴毒素β受体。 免疫力 42(6):1100-1115。
  10. Kyaw,T.,Tay,C.,Hosseini,H.,Kanellakis,P.,Gadowski,T.,MacKay,F.,Tipping,P.,Bobik,A。和Toh,BH(2012)在BAFF受体缺陷型ApoE小鼠中,B2而不是B1a B细胞的消耗量减少了,这是因为:a ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22238605"target ="_ blank"通过有效改善动脉炎症来减弱动脉粥样硬化。 7(1):e29371。
  11. Lotzer,K.,Dopping,S.,Connert,S.,Grabner,R.,Spanbroek,R.,Lemser,B.,Beer,M.,Hildner,M.,Hehlgans,T.,van der Wall,M 。,Mebius,RE,Lovas,A.,Randolph,GJ,Weih,F。和Habenicht,AJ(2010)。 小鼠主动脉平滑肌细胞分化为淋巴组织样组织细胞联合肿瘤坏死因子受体-1 /淋巴毒素β受体NF-κB信号。 Arterioscler Thromb Vasc Biol 30(3):395-402。
  12. Mohanta,S.,Yin,C.,Weber,C.,Hu,D.and Habenicht,AJR(2016)。  高脂血症小鼠中的主动脉粥样硬化病变分析。生物方案 6(11):e1833。
  13. Mohanta,SK,Yin,C.,Peng,L.,Srikakulapu,P.,Bontha,V.,Hu,D.,Weih,F.,Weber,C.,Gerdes,N.and Habenicht,AJ(2014) 。 动脉三级淋巴器官促进先天小鼠的先天性和适应性免疫反应动脉粥样硬化。 Circ Res 114(11):1772-1787。
  14. Moos,MP,John,N.,Grabner,R.,Nossmann,S.,Gunther,B.,Vollandt,R.,Funk,CD,Kaiser,B.and Habenicht,AJ(2005) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16179593"target ="_ blank">椎板外膜是标准食物载脂蛋白载脂蛋白中免疫细胞积累的主要部位E缺陷小鼠。 Arterioscler Thromb Vasc Biol 25(11):2386-2391。
  15. Srikakulapu,P.,Hu,D.,Yin,C.,Mohanta,SK,Vineela Bontha,S.,Peng,L.,Beer,M.,Weber,C.,McNamara,CA,Grassia,G.,Maffia ,P.,Manz,RA和Habenicht,AJ(2016)。  动脉三级淋巴器官控制多层领域动脉粥样硬化在老年ApoE ITALIC的B细胞反应! -/- 小鼠。 Arterioscler Thromb Vasc Biol 。
  16. Zhao,L.,Moos,MP,Grabner,R.,Pedrono,F.,Fan,J.,Kaiser,B.,John,N.,Schmidt,S.,Spanbroek,R.,Lotzer, ,L.,Cui,J.,Rader,DJ,Evans,JF,Habenicht,AJand Funk,CD(2004)。 5-脂氧合酶途径促进高脂血症依赖性主动脉瘤的发病机制。 Nat Med 10(9):966-973。
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引用:Hu, D., Yin, C., Mohanta, S., Weber, C. and Habenicht, A. J. (2016). Preparation of Single Cell Suspensions from Mouse Aorta. Bio-protocol 6(11): e1832. DOI: 10.21769/BioProtoc.1832.
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