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Adoptive Transfer of Lung Antigen Presenting Cells
肺抗原呈递细胞的过继转移   

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Abstract

Our protocol describes adoptive transfer of antigen presenting cells (APCs) isolated from the lungs by enzymatic digestion and magnetic enrichment. This protocol can be used to study APC functions and trafficking.

Keywords: Lung antigen presenting cells(肺抗原呈递细胞), Dendritic cells(树突状细胞), Macrophages(巨噬细胞), Adoptive transfer(过继转移), Enzymatic digestion(酶消化), Magnetic cell isolation(磁性细胞分离), Intravenous injection(静脉注射)

Background

Lung APCs, including macrophages and dendritic cells (DCs), play a critical role in sensing invading pathogens, priming T cell responses and controlling tolerogenic responses. Lung DCs are the most potent professional APCs, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) during steady-state, and newly recruited monocyte-derived DCs (moDCs) upon inflammation (Kopf et al., 2015). Lung resident macrophages, including alveolar macrophages, interstitial macrophages and bronchial macrophages, are less potent in presenting antigens.

All macrophages and cDCs express high levels of CD11c on their cell surface, while pDCs express intermediate levels of CD11c (Becher et al., 2014). Thus, CD11c magnetic microbeads can be used to isolate mouse lung macrophages and cDCs. We developed a protocol of isolating lung APCs from normal mice and then adoptively transferring them to syngeneic bone marrow transplanted mice. We used this protocol to determine whether normal lung APCs are sufficient to restore T helper cell polarization in response to herpesvirus infection post-transplant (Zhou et al., 2016).

Materials and Reagents

  1. PrecisionGlide needles 23 G (BD, catalog number: 305111 )
  2. PrecisionGlide 26 G (BD, catalog number: 305193 )
  3. 60 x 15 mm Petri dish (Corning, Falcon®, catalog number: 351007 )
  4. 50 ml flip-top conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 362696 )
  5. 10 ml Luer slip tip syringe (Fisher Scientific, catalog number: 14-841-55 )
  6. Razor blade
  7. Tissue culture plates
  8. 100 µm nylon screen (Nylon Mesh Lab Pack) (Sefar, catalog number: 7050-1220-000-20 )
  9. MACS LS column (Miltenyi Biotec, catalog number: 130-042-401 )
  10. 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  11. 1 ml slip tip syringe (BD, catalog number: 309659 )
  12. 0.22 µm filter (Millipore Express PLUS 0.22 µm PES) (EMD Millipore, catalog number: SCGPU01RE )
  13. Pipette tips  
  14. Latex gloves
  15. Syringe tip caps (BD, catalog number: 305819 )
  16. Mice
  17. 70% ethanol
  18. Phosphate buffered saline (PBS, pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  19. Heat-inactivated fetal bovine serum (Mediatech, catalog number: 35-010-CV )
  20. Penicillin-streptomycin 100x (Mediatech, catalog number: 30-002-CI )
  21. L-glutamine 100x, 200 mM (Thermo Fisher Scientific, GibcoTM, 25030-018 )
  22. Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  23. Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, catalog number: 12-604F )
  24. Collagenase A (Roche Diagnostics, catalog number: 10103578001 )
  25. DNase I (Sigma-Aldrich, catalog number: D4263 )
  26. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 254134 )
  27. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 431583 )
  28. EDTA, 0.5 M (Lonza, catalog number: 51201 )
  29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
  30. Percoll (Sigma-Aldrich, catalog number: P1644 )
  31. Mouse CD11c MicroBeads UltraPure (Miltenyi Biotec, catalog number: 130-108-338 )
  32. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  33. Complete media (see Recipes)
  34. Collagenase digest solution (see Recipes)
  35. RBC lysis buffer (see Recipes)
  36. Serum free media (see Recipes)
  37. MACS buffer (see Recipes)

Equipment

  1. CO2 tank
  2. Scissors
  3. Forceps
  4. Water bath
  5. Cell culture incubator
  6. Rocker
  7. Refrigerated Swing bucket centrifuge (Beckman Coulter, model: Allegra X-12R )
  8. Vortex
  9. Hemocytometer
  10. Mouse restrainer
  11. Heat lamp
  12. Pipette  
  13. Compound microscope
  14. MACS separator (Miltenyi Biotec)

Procedure

  1. Preparing single cell suspension from the lungs of donor mice
    1. Euthanize mice with CO2. Cut the rib cage to expose both the heart and lungs (Figures 1A and 1B).
    2. To remove blood cells from the lung, perfuse the lungs with 3-5 ml PBS using a 23 gauge needle via the right ventricle of the heart (Figure 1C). Gently inject PBS till the color of lungs turns to white. Note that redness may maintain throughout perfusion in areas inflamed or injured.
    3. Excise the lung lobes (total 5 per mouse, Figure 1D) and place them in a 60 x 15 mm Petri dish with approximately 1 ml cold PBS and store on ice until all the lungs are harvested from all the mice.
    4. Remove lungs from PBS and mince with scissors in the lid of the Petri dish until the consistency of applesauce; keep lungs from each mouse separate (Figure 1E).


      Figure 1. Preparing lung tissue. A. Pin a euthanized mouse on a plastic foam board. B. Cut the rib cage to expose both the heart (H) and the lungs (L). C. Perfuse the lungs by injecting PBS into the right ventricle of the heart. The black arrow points to the needle inserting into the heart. D. Lung lobes (5) are removed and placed in a Petri dish with cold PBS. E. The lungs are minced.

    5. Rinse the scissors and the Petri dish with 15 ml collagenase digest solution (see Recipes below) and place into a 50 ml conical centrifuge tube.
    6. Close the tubes and incubate the tubes for 5 min in a 37 °C water bath and then for 30 min in a 37 °C incubator on a rocker.
    7. Using a 10 ml slip tip syringe, draw the sample up and down 20 times to disperse cells.
    8. Centrifuge for 10 min at 200 x g (i.e., 1,000 rpm) in a swing bucket centrifuge.
    9. Aspirate the supernatant and discard; break up the pellet by vortexing gently.
    10. Add 3 ml cold RBC lysis buffer (NH4Cl, see Recipes below) per sample to lyse red blood cells, incubate on ice 2-3 min.
    11. Add 10 ml cold complete media (see Recipes) to each tube to stop lysis and pellet again at 200 x g for 10 min at 4 °C.
    12. Aspirate supernatant, break up the pellet, and add 5 ml serum-free media (see Recipes) to resuspend the cells.
    13. Cut tip off syringe tip cap with razor blade and place it on a 10 ml slip tip syringe (this is to make the syringe tip a little longer for dispersing the volume smaller than 5 ml. Draw the sample up and down 20 times to disperse cells.
    14. Draw up the solution into the syringe and filter through a sterile 100 µm nylon screen. Rinse the screen with an additional 5 ml of complete media.
    15. Add 10 ml 40% Percoll in complete media. Mix well by inverting the tubes 5 times and centrifuge for 20 min at 1,800 x g (i.e., 3,000 rpm), turn off brake.
    16. Aspirate supernatant and discard; break up pellet with 10 ml of complete media. Count cells with trypan blue exclusion on a hemocytometer.
      Note: We usually recover 16-17 million cells/naïve mouse.

  2. Lung APC enrichment
    Note: In our hands, the yield of lung APCs is usually about 5% of total cells in the lung single cell suspension. One may roughly calculate a starting total lung cell number in order to obtain enough lung APCs for his/her experiments.
    1. Centrifuge single cell suspension for 10 min at 200 x g and aspirate supernatant and discard.
    2. Resuspend cell pellet in 400 µl of cold MACS buffer (see Recipes) for less than or equal to 108 total lung cells and add 100 µl of CD11c MicroBeads. Scale up buffer and beads if more than 108 total cells.
    3. Mix well and incubate for 15 min in the refrigerator (2-8 °C). Note that working on ice may require a longer incubation time.
    4. Wash cells with 10 ml cold MACS buffer per 108 cells and centrifuge at 200 x g for 10 min at 4 °C.
    5. Aspirate supernatant and then resuspend cell pellet in 500 µl of MACS buffer.
    6. Place column in the magnetic field of a MACS LS column. Prepare column by rinsing with 3 ml MACS buffer.
    7. Place a sterile 100 µm nylon screen on the column. Apply cell suspension onto the column by filtering through the nylon screen. Collect flow-through containing unlabeled cells in a 15 ml conical centrifuge tube.
    8. Wash column with 3 ml of MACs buffer and collect unlabeled flow-through cells. Wash 3 times.
    9. Remove column from the separator and place it over a new 15 ml conical centrifuge tube.
    10. Pipette 5 ml of buffer on the column. Immediately flush out the magnetically labelled cells by firmly pushing the plunger into the column.
    11. Centrifuge enriched CD11c+ cells at 200 x g for 10 min and discard supernatant.
    12. Wash cells by gently breaking up the pellet with 10 ml serum free medium. Count cells with trypan blue exclusion on a hemocytometer. Note, usually recover 1-2 million APCs/mouse.
    13. Centrifuge at 200 x g for 10 min and discard supernatant. Resuspend cells in appropriate volume of serum-free medium to reach a final concentration of 1 x 106 cells per 200 µl.

  3. Adoptive transfer of lung APCs into recipients via intravenous injection
    Note: Adoptive transfer can also be achieved by intratracheal administration (for detailed protocol of intratracheal administration, please refer to Ortiz-Muñoz and Looney, 2015, Bio-protocol).
    1. Load cell suspension into a 1 ml syringe with 26 G needle. Make sure to remove air bubbles.
    2. Place recipient mouse in a plastic mouse restrainer and warm the tail with a heat lamp for about one minute (Figure 2A).
    3. Immobilize the tail with gentle traction (Figure 2A).
    4. Spray the tail with 70% ethanol to visualize the lateral tail vein and rotate the tail ¼ turn for easier injection.
    5. At the distal portion of the tail, insert needle parallel to the vein 2 to 5 mm into to the lumen (Figure 2B).
    6. Inject 1 x 106 cells in 200 µl per mouse. Inject the cell suspension slowly. The cell suspension should flow easily if needle is properly located.


      Figure 2. Tail vein injection. A. A recipient mouse is placed in a restrainer. B. Insert a needle into a mouse tail vein to inject cells.

    7. Withdraw the needle and apply pressure to prevent bleeding.
    8. Place the recipient mouse back into its cage.
    9. Mice may then receive designated treatments and can be analyzed at appropriate time points.

Data analysis

Single cell suspension was prepared from the lungs of C57BL/6 mice three days post infection of murine gamma herpesvirus 68 (MHV-68) intranasal at a dose of 5 x 104 pfu/mouse (Zhou et al., 2016). CD11c+ cells were then enriched by using anti-mouse CD11c microbeads. To analyze the composition of these microbead-enriched cells by flow cytometry, appropriate fluorescence-conjugated antibodies were used to stain cell surface markers. Percentages on each plot represent the percentages of each parental gate. Similar results were obtained in two additional independent experiments. The majority enriched CD11c+ APCs are alveolar macrophages (AMs) (CD45+ CD11c+ MHC IIint CD64hi), and DCs. The DC population can be further separated into CD103 cDCs (CD45+ CD11c+ MHC IIhi CD64- CD103hi CD11bint), CD11b+ cDCs (CD45+ CD11c+ MHC IIhi CD64- CD103lo CD11bhi) and MoDCs (CD45+ CD11c+ MHC IIhi CD64+ CD103lo CD11bhi) (Misharin et al., 2013; Wang et al., 2006) (Figure 3) .
Lung APCs were prepared from congenic CD45.1 mice three days post infection of MHV-68, and adoptively transferred into wild-type C57BL/6 mice (CD45.2+, but CD45.1-). The recipients were infected with MHV-68 24 h after cell transfer, and euthanized for flow cytometry analysis 48 h after cell transfer. Representative flow cytometry plots show donor CD45.1+ APCs in the lungs, lung draining lymph nodes (dLN) and spleens of CD45.1- recipients (Figure 4).


Figure 3. Representative flow cytometry plots of APCs isolated from mouse lungs


Figure 4. Tracking injected cells. Upper panel, wild-type C57BL/6 mice without cell transfer; lower panel, wild-type C57BL/6 recipients adoptively transferred with CD45.1+ lung APCs.

Notes

If CD11c+ subpopulations are under investigation, the magnetically enriched cells can be further stained with appropriate fluorescence-conjugated antibodies and sorted to specific APC populations using fluorescence-activated cell sorting (FACS) (Misharin et al., 2013).

Recipes

  1. Complete media
    Mix the following solutions and filter through 0.22 µm filter:
    50 ml heat-inactivated fetal bovine serum
    5 ml 100x penicillin-streptomycin (10,000 IU penicillin and 10,000 µg/ml streptomycin)
    5 ml 100x L-glutamine (200 mM)
    0.5 ml amphotericin B (250 µg/ml)
    Add the filtered mixture into a 500 ml bottle of DMEM media
  2. Collagenase digest solution (per mouse, prepare immediately before use)
    15 mg collagenase A
    0.25 ml DNase I (250 Kunitz unit)
    15 ml complete media
  3. RBC lysis buffer
    Dissolve the following in 800 ml distilled H2O
    8.3 g NH4Cl
    1.0 g KHCO3
    1.8 ml of 5% EDTA
    Add distilled H2O up to a total volume of 1,000 ml
    Filter sterilize through a 0.22 µm filter, and keep in refrigerator
  4. Serum free media
    500 ml DMEM media
    5 ml 10% BSA
    5 ml 100x penicillin-streptomycin (10,000 IU penicillin and 10,000 µg/ml streptomycin)
    5 ml 100x L-glutamine (200 mM)
    0.5 ml amphotericin B (250 µg/ml)
    Store in refrigerator (4-8 °C)
  5. MACS buffer
    For 150 ml solution:
    7.5 ml of 10% bovine serum albumin (BSA)
    0.6 ml of 0.5 M EDTA
    Store in refrigerator (4-8 °C)

Acknowledgments

This protocol was adapted from our publication (Zhou et al., 2016). This work was supported by NIH grants AI117229, HL115618, T32HL07749 and 2UL1TR000433.

References

  1. Becher, B., Schlitzer, A., Chen, J., Mair, F., Sumatoh, H. R., Teng, K. W., Low, D., Ruedl, C., Riccardi-Castagnoli, P., Poidinger, M., Greter, M., Ginhoux, F. and Newell, E. W. (2014). High-dimensional analysis of the murine myeloid cell system. Nat Immunol 15(12): 1181-1189.
  2. Kopf, M., Schneider, C. and Nobs, S. P. (2015). The development and function of lung-resident macrophages and dendritic cells. Nat Immunol 16(1): 36-44.
  3. Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R. and Perlman, H. (2013). Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. Am J Respir Cell Mol Biol 49(4): 503-510.
  4. Ortiz-Muñoz, G. and Looney, M. R. (2015). Non-invasive intratracheal instillation in mice. Bio-protocol 5(12): e1504.
  5. Wang, H., Peters, N., Laza-Stanca, V., Nawroly, N., Johnston, S. L. and Schwarze, J. (2006). Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process. J Immunol 177(4): 2536-2542.
  6. Zhou, X., Loomis-King, H., Gurczynski, S. J., Wilke, C. A., Konopka, K. E., Ptaschinski, C., Coomes, S. M., Iwakura, Y., van Dyk, L. F., Lukacs, N. W. and Moore, B. B. (2016). Bone marrow transplantation alters lung antigen-presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection. Mucosal Immunol 9(3): 610-620.

简介

我们的方案描述了通过酶消化和磁力富集从肺分离的抗原呈递细胞(APC)的过继转移。该协议可用于研究APC功能和贩运。

背景 包括巨噬细胞和树突状细胞(DC)在内的肺癌APC在感染侵袭性病原体,引发T细胞应答和控制耐受性反应中发挥关键作用。肺DC是稳定状态下最有效的专业APC,包括常规DC(cDC)和浆细胞样DC(pDC),以及炎症时新招募的单核细胞衍生的DC(moDC)(Kopf等人)。 ,2015)。肺静脉巨噬细胞,包括肺泡巨噬细胞,间质巨噬细胞和支气管巨噬细胞,在呈递抗原方面的效力较低。
 所有巨噬细胞和cDC在其细胞表面表达高水平的CD11c,而pDC表达中等水平的CD11c(Becher等,2014)。因此,CD11c磁性微珠可用于分离小鼠肺巨噬细胞和cDC。我们开发了一种从正常小鼠中分离肺APCs的方案,然后将其过渡转移到同基因骨髓移植小鼠。我们使用该方案来确定正常的肺APCs是否足以恢复移植后疱疹病毒感染的T辅助细胞极化(Zhou等人,2016)。

关键字:肺抗原呈递细胞, 树突状细胞, 巨噬细胞, 过继转移, 酶消化, 磁性细胞分离, 静脉注射

材料和试剂

  1. PrecisionGlide针头23 G(BD,目录号:305111)
  2. PrecisionGlide 26 G(BD,目录号:305193)
  3. 60×15mm培养皿(Corning,Falcon ®,目录号:351007)
  4. 50ml旋转锥形离心管(Thermo Fisher Scientific,目录号:362696)
  5. 10ml Luer滑点注射器(Fisher Scientific,目录号:14-841-55)
  6. 剃刀刀片
  7. 组织培养板
  8. 100微米尼龙丝网(尼龙网实验室包装)(Sefar,目录号:7050-1220-000-20)
  9. MACS LS栏(Miltenyi Biotec,目录号:130-042-401)
  10. 15ml锥形离心管(Corning,Falcon ®,目录号:352097)
  11. 1毫升滑点注射器(BD,目录号:309659)
  12. 0.22μm过滤器(Millipore Express PLUS0.22μmPES)(EMD Millipore,目录号:SCGPU01RE)
  13. 移液器提示
  14. 乳胶手套
  15. 注射器盖帽(BD,目录号:305819)
  16. 小鼠
  17. 70%乙醇
  18. 磷酸盐缓冲盐水(PBS,pH 7.4)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  19. 热灭活的胎牛血清(Mediatech,目录号:35-010-CV)
  20. 青霉素 - 链霉素100x(Mediatech,目录号:30-002-CI)
  21. L-谷氨酰胺100x,200mM(Thermo Fisher Scientific,Gibco TM,25030-018)
  22. 两性霉素B(Thermo Fisher Scientific,Gibco TM ,目录号:15290018)
  23. Dulbecco改良Eagle's培养基(DMEM)(Lonza,目录号:12-604F)
  24. 胶原酶A(Roche Diagnostics,目录号:10103578001)
  25. DNase I(Sigma-Aldrich,目录号:D4263)
  26. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:254134)
  27. 碳酸氢钾(KHCO 3)(Sigma-Aldrich,目录号:431583)
  28. EDTA,0.5M(Lonza,目录号:51201)
  29. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912)
  30. Percoll(Sigma-Aldrich,目录号:P1644)
  31. 鼠标CD11c MicroBeads UltraPure(Miltenyi Biotec,目录号:130-108-338)
  32. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  33. 完整的媒体(见配方)
  34. 胶原酶消化液(参见食谱)
  35. RBC裂解缓冲液(参见食谱)
  36. 无血清培养基(见食谱)
  37. MACS缓冲区(见配方)

设备

  1. CO 2 tank
  2. 剪刀
  3. 镊子
  4. 水浴
  5. 细胞培养箱
  6. 摇杆
  7. 冷藏摇摆式离心机(Beckman Coulter,型号:Allegra X-12R)
  8. 涡流
  9. 血细胞计数器
  10. 鼠标限制器
  11. 加热灯
  12. 移液器
  13. 复合显微镜
  14. MACS分隔符(Miltenyi Biotec)

程序

  1. 从供体小鼠的肺中制备单细胞悬液
    1. 用CO 2安乐死小鼠。切割肋骨以暴露心脏和肺部(图1A和1B)。
    2. 为了从肺中清除血细胞,使用23号针头通过心脏的右心室,用3-5ml PBS灌注肺(图1C)。轻轻注射PBS直到肺的颜色变成白色。请注意,发炎或受伤的地区,灌注发生红斑。
    3. 消除肺叶(总共5只,每只小鼠,图1D),并将它们放在一个60 x 15毫米的陪替氏培养皿中,约1毫升冷PBS,并储存在冰上,直到所有的小鼠都收获所有的肺。
    4. 从PBS中取出肺,用剪刀将培养皿盖在培养皿的盖子上,直到苹果酱的一致性;保持每只小鼠的肺分开(图1E)

      图1.准备肺组织 A.将安乐死的小鼠放在塑料泡沫板上。 B.切割肋骨以暴露心脏(H)和肺(L)。 C.通过将PBS注射到心脏的右心室来灌注肺。黑色箭头指向插入心脏的针头。 D.将肺叶(5)取出并放入含冷PBS的培养皿中。 E.肺切碎。

    5. 用15ml胶原酶消化液冲洗剪刀和培养皿(参见下面的配方),并放入50ml圆锥形离心管中。
    6. 关闭管,并在37℃水浴中孵育管5分钟,然后在37℃的摇床上孵育30分钟。
    7. 使用10ml滑点注射器,上下抽取样品20次以分散细胞。
    8. 在摆动式离心机中以200×g离心10分钟(即1,000rpm)离心10分钟。
    9. 吸出上清液并弃去;通过轻轻涡旋分解沉淀物
    10. 每个样品加入3 ml冷RBC裂解缓冲液(NH 4 Cl,见下面的配方)以裂解红细胞,在冰上孵育2-3分钟。
    11. 向每个管中加入10ml冷的完整培养基(参见食谱)以在4℃下以200×g×10分钟再次停止裂解并沉淀。
    12. 吸出上清液,分离沉淀,加入5ml无血清培养基(参见食谱)重悬细胞。
    13. 用剃须刀切开尖头,将其放在10毫升滑点注射器上(这是为了使注射器针头稍长一点,以便分散体积小于5毫升。将样品上下抽取20次以分散细胞。
    14. 将溶液吸入注射器,并通过无菌的100μm尼龙筛网进行过滤。用另外5毫升完整的介质冲洗屏幕。
    15. 在完整培养基中加入10 ml 40%Percoll。通过将管倒置5次并以1,800 x g(即,3,000rpm)离心20分钟,将制动器关掉。
    16. 吸出上清液并弃去;用10ml完整培养基分解沉淀。在血细胞计数器上计数细胞与台盼蓝排除。
      注意:我们通常会恢复16-17万个细胞/天真的老鼠。

  2. 肺APC浓缩
    注意:在我们手中,肺APCs的产量通常为肺单细胞悬液中总细胞的5%。人们可以粗略地计算起始的总肺细胞数,以获得足够的肺APC用于他/她的实验。
    1. 在200×g离心单细胞悬浮液10分钟并吸出上清液并弃去。
    2. 将细胞沉淀重悬于400μl冷MACS缓冲液(参见食谱)中,小于或等于10μg/ml总肺细胞,并加入100μlCD11c MicroBeads。如果超过10个总细胞,请放大缓冲液和珠子。
    3. 混合均匀,并在冰箱(2-8℃)中孵育15分钟。请注意,在冰上工作可能需要较长的孵化时间。
    4. 用10ml冷MACS缓冲液/10 8细胞洗涤细胞,并在4℃以200×g离心10分钟。
    5. 吸出上清,然后将细胞沉淀重悬于500μlMACS缓冲液中。
    6. 将列放在MACS LS列的磁场中。用3ml MACS缓冲液冲洗准备柱。
    7. 在柱上放置无菌的100μm尼龙筛网。通过尼龙筛网过滤,将细胞悬浮液施加到柱上。在15 ml锥形离心管中收集含有未标记细胞的流通。
    8. 用3ml MAC缓冲液洗涤柱并收集未标记的流通池。洗3次。
    9. 从分离器中取出柱,并将其放在新的15 ml锥形离心管上。
    10. 移取柱上的5ml缓冲液。通过将柱塞牢固地推入柱子中,立即将磁性标记的细胞冲洗掉。
    11. 将离心浓缩的CD11c + 细胞在200×g下10分钟,弃去上清液。
    12. 通过用10ml无血清培养基轻轻分解沉淀物来洗涤细胞。在血细胞计数器上计数细胞与台盼蓝排除。注意,通常恢复1-2百万APC /鼠标。
    13. 以200μg离心10分钟并弃去上清液。将细胞重悬于适当体积的无血清培养基中,以达到每200μl终浓度为1×10 6个细胞。

  3. 通过静脉内注射将肺APCs转移到接受者中 注意:通过气管内给药也可以实现继发性转移(气管内给药的详细方案请参见Ortiz-Muñoz和Looney,2015,Bio-protocol)。
    1. 将称重传感器悬浮液装入带有26 G针头的1 ml注射器中。确保清除气泡。
    2. 将收件人的鼠标放在塑料鼠标限制器中,并用加热灯将尾巴加热约1分钟(图2A)。
    3. 用温和的牵引力固定尾巴(图2A)。
    4. 用70%乙醇喷洒尾巴以显示侧尾静脉,并旋转尾巴¼转,便于注射。
    5. 在尾部的远端部分,将平行于静脉的针2〜5mm插入内腔(图2B)。
    6. 以每只小鼠200μl注射1×10 6个细胞。缓慢注射细胞悬浮液。如果针头正确定位,细胞悬液应该容易流动。


      图2.尾静脉注射 A.将受体鼠标放置在限制器中。 B.将针头插入小鼠尾静脉注射细胞
    7. 取出针头并施加压力以防止出血。
    8. 将收件人的老鼠放回笼子里。
    9. 然后,小鼠可以接受指定的治疗,并可在适当的时间点进行分析。

数据分析

在5×10 4 pfu /小鼠的剂量下感染鼠γ疱疹病毒68(MHV-68)三天后,从C57BL/6小鼠的肺制备单细胞悬液(Zhou, em> et al。 2016)。然后通过使用抗小鼠CD11c微珠来浓缩CD11c + 细胞。为了通过流式细胞术分析这些富含微细胞的细胞的组成,使用合适的荧光偶联抗体染色细胞表面标志物。每个地块的百分比代表每个家长门的百分比。在两次额外的独立实验中获得了类似的结果。大多数富集的CD11c + APCs是肺泡巨噬细胞(AM)(CD45 + CD11c +//> MHC II sup> hi )和DC。 DC群体可以进一步分离成CD103cDC(CD45cCDC),CD11c +//> MHC II CD64 (CD11b int ),CD11b + cDCs(CD45 + CD11c + MHC II < (CDCD) CD103 CD103 CD103 > + MHC II CD64 + CD103 lo CD11b hi )(Misharin et al。 ,2013; Wang等人,2006)(图3)。
在MHV-68感染后3天,从同源CD45.1小鼠制备肺APCs,并过继转移到野生型C57BL/6小鼠(CD45.2,CD45.1, - )。接受者在细胞转移后24小时感染MHV-68,并在细胞转移48小时后安乐死用于流式细胞术分析。代表性的流式细胞术图显示供体CD45.1 + APCs在肺,肺引流淋巴结(dLN)和CD45.1 接受者的脾脏(图4)。 br />

图3.从小鼠肺分离的APC的代表性流式细胞术图


图4.跟踪注入的细胞。上面,没有细胞转移的野生型C57BL/6小鼠;下盘,野生型C57BL/6受体通过CD45.1 肺APC继续转移。

笔记

如果CD11c + 亚群正在研究中,则可以用合适的荧光结合抗体进一步染色磁性富集的细胞,并使用荧光激活细胞分选(FACS)(Misharin et al。 al 。,2013)。

食谱

  1. 完成媒体
    混合以下溶液并通过0.22μm过滤器过滤:
    50ml热灭活胎牛血清 5 ml 100x青霉素 - 链霉素(10,000 IU青霉素和10,000μg/ml链霉素)
    5ml 100x L-谷氨酰胺(200mM)
    0.5ml两性霉素B(250μg/ml)
    将过滤后的混合物加入到500ml DMEM培养基中
  2. 胶原酶消化液(每只鼠,使用前立即准备)
    15毫克胶原酶A
    0.25 ml DNase I(250 Kunitz单位)
    15毫升完整媒体
  3. RBC裂解缓冲液
    将以下物质溶解在800ml蒸馏水中的二氧化O O O。。 8.3g NH 4 Cl
    1.0克KHCO 3
    1.8ml 5%EDTA
    加入蒸馏的H 2 O 2,总体积为1000ml
    过滤器通过0.22μm过滤器灭菌,并保存在冰箱中
  4. 血清免费媒体
    500毫升DMEM培养基
    5 ml 10%BSA
    5 ml 100x青霉素 - 链霉素(10,000 IU青霉素和10,000μg/ml链霉素)
    5ml 100x L-谷氨酰胺(200mM)
    0.5ml两性霉素B(250μg/ml)
    储存在冰箱(4-8°C)
  5. MACS缓冲区
    150 ml溶液:
    7.5毫升10%牛血清白蛋白(BSA)
    0.6ml 0.5M EDTA 储存在冰箱(4-8°C)

致谢

这个协议是从我们的出版物(Zhou等人,2016)改编而成。这项工作得到NIH授权AI117229,HL115618,T32HL07749和2UL1TR000433的支持。

参考文献

  1. Becher,B.,Schlitzer,A.,Chen,J.,Mair,F.,Sumatoh,HR,Teng,KW,Low,D.,Ruedl,C.,Riccardi-Castagnoli,P.,Poidinger, Greter,M.,Ginhoux,F.and Newell,EW(2014)。鼠骨髓细胞系统的高维分析。 Nat Immunol 15(12):1181-1189。
  2. Kopf,M.,Schneider,C.and Nobs,SP(2015)。  肺驻极体巨噬细胞和树突状细胞的发展和功能。免疫学杂志 16(1):36-44。
  3. Misharin,AV,Morales-Nebreda,L.,Mutlu,GM,Budinger,GR和Perlman,H。(2013)。  小鼠肺中巨噬细胞和树突状细胞亚群的流式细胞术分析。 Am J Respir Cell Mol Biol 49(4) :503-510。
  4. Ortiz-Muñoz,G. and Looney,MR(2015)。  小鼠无创气管内滴注。生物方案5(12):e1504。
  5. Wang,H.,Peters,N.,Laza-Stanca,V.,Nawroly,N.,Johnston,SL and Schwarze,J。(2006)。  本地CD11c + MHC class II - 前体在呼吸过程中产生肺树突状细胞病毒感染,但在此过程中耗尽。 J Immunol 177(4):2536-2542。
  6. Zhou,X.,Loomis-King,H.,Gurczynski,SJ,Wilke,CA,Konopka,KE,Ptaschinski,C.,Coomes,SM,Iwakura,Y.,van Dyk,LF,Lukacs,NW和Moore,BB (2016)。骨髓移植改变肺抗原呈递细胞促进TH17反应,以及伽pes病毒感染后肺炎和纤维化的发展。粘膜免疫学9(3):610-620。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhou, X. and Moore, B. B. (2017). Adoptive Transfer of Lung Antigen Presenting Cells. Bio-protocol 7(6): e2182. DOI: 10.21769/BioProtoc.2182.
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