搜索

C1q Binding to and Uptake of Apoptotic Lymphocytes by Human Monocyte-derived Macrophages
通过人单核细胞源巨噬细胞进行凋亡淋巴细胞的Clq结合和吞噬

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

To characterize macrophage gene expression profiles during the uptake of autologous apoptotic cells, we developed a unique, more physiologic system using primary human monocyte derived macrophages purified via a nonactivating isolation procedure (and in the absence of contaminating platelets, which can release stimulating signals if activated) and autologous lymphocytes as a source of apoptotic cells. The use of autologous cells as the apoptotic target rather than transformed cell lines avoids antigenic stimulation from “nonself” structures at the HLA level but also from “altered self” signals due to the transformation inherent in cell lines.

Keywords: Apoptotic cell clearance(凋亡细胞的清除), Autoimmunity(自身免疫性疾病), Macrophages(巨噬细胞), Human(人类), C1q(C1q)

Material and Reagents

  1. Human peripheral blood
  2. RPMI1640 + L-Glutamine + HEPES (Life Technologies, catalog number: 22400-105 )
  3. FBS (inactivate for 30 min at 56 °C) (Hyclone defined FBS, catalog number: SH30070.03 )
  4. Penicillin/Streptomycin (Life Technologies, catalog number: 15070-063 )
  5. L-Glutamine (200 mM) (Life Technologies, catalog number: 25030-081 )
  6. 0.4% Trypan blue Solution (Sigma-Aldrich, catalog number: T8154 )
  7. HBSS (Corning/Cellgro, catalog number: MT-21-023-CV )
  8. PBS
  9. 25% Human Serum Albumin (HSA) (Plasbumin®-25) (Talecris Biotherapeutics, NDC number: 13533-684-20 )
  10. Recombinant human IL-2 (Peprotech, catalog number: 200-02 )
  11. Recombinant human M-CSF (Peprotech, catalog number: 300-25 )
  12. Bovine Serum Albumin (BSA) (albumin from bovine serum, lyophilized powder, ≥ 96%) (Sigma-Aldrich, catalog number: A2153-100G )
  13. C1q (Comptech, Texas, catalog number: A099 )
  14. PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich, catalog number: PKH26GL )
  15. CellStripper Dissociation Reagent (Thermo Fisher Scientific, catalog number: 25-056-CI )
  16. Apoptosis detection kit (BioVision, catalog number: K101-100 )
  17. Anti-human C1q (Quidel, catalog number: A201 )
  18. FITC conjugated anti-mouse IgG (Jackson Immunoresearch, catalog number: 115-096-006 )
  19. FcR blocking reagent human (Miltenyi, catalog number: 130-059-901 )
  20. FITC conjugated anti-human CD11b (Life Technologies, catalog number: CD11B01 )
  21. FITC mouse IgG1 isotypes (Life Technologies, catalog number: MG101 )
  22. Fluorescein phalloidin (Life Technologies, catalog number: F432 )
  23. Stericup (Thermo Fisher Scientific, catalog number: SCGVU11RE )
  24. Prolong gold antifade reagent (Life Technologies, catalog number: P36930 )
  25. Ammonium Chloride (anhydrous)
  26. Potassium bicarbonate
  27. Disodium EDTA
  28. NaN3
  29. Trypsin
  30. Complete media (see Recipes)
  31. Phagocytosis buffer (see Recipes)
  32. ACK buffer (see Recipes)
  33. FACS buffer (see Recipes)

Equipment

  1. 50 ml conical tubes (Thermo Fisher Scientific, catalog number: 339653)–important as monocytes stick to other tubes
  2. 12 x 75 mm polypropylene round bottom sterile tube (Thermo Fisher Scientific, catalog number: 14-956-1D )
  3. 12 x 75 mm polystyrene round bottom tubes (Thermo Fisher Scientific, catalog number: 14-961-13 )
  4. Tissue culture plates and vented flasks (any size)
  5. Eppendorf microfuge tubes
  6. 12 mm cover slips (Thermo Fisher Scientific, catalog number: GG12 ), sterilize by soaking in 70% Ethanol for 2 x 5 min
  7. Non tissue culture treated 100 mm petri dish (Thermo Fisher Scientific, catalog number: 0875712 )–referred to as petri dishes
  8. Tissue culture hood
  9. Gamma-irradiator
  10. 5% CO2, 37 °C Humidified incubator
  11. Centrifuge for 50 ml conical tubes and 5 ml bottom round tubes
  12. Centrifuge with swinging bucket rotor for plates (Sorvall, model: RT7000 or equivalent)
  13. Optic and confocal fluorescence microscopes
  14. Hemocytometer
  15. Flow cytometer
  16. Automatic pipettes (full range volumes)
  17. Tips (full range volumes)
  18. 24-well plates

Procedure


  1. Lymphocyte isolation, staining and apoptosis induction.
    1. Collect the first two 50 ml effluent tubes from the elutriation, which contain lymphocytes (monocytes are retained in the elutriation chamber).
    2. Centrifuge lymphocyte suspension 10 min at 700 rpm (100 x g), RT (room temperature) to remove majority of platelets.
    3. Discard supernatant and pool cell pellets in 10 ml ACK buffer (to remove residual red cells). Incubate 2-5 min RT.
    4. Add 20 ml complete media.
    5. Centrifuge cell suspension 10 min at 700 rpm (100 x g), RT.
    6. Discard supernatant and resuspend the cell pellet in 20 ml HBSS. Count viable cell number using 0.4% Trypan blue solution, a hemocytometer chamber and an optic microscope.
    7. Centrifuge cell suspension 10 min at 700 rpm (100 x g), RT.
    8. Resuspend the lymphocytes at 1 million/ml in complete media in a vented tissue culture flask.
    9. Add 100 U/ml recombinant human IL-2.
    10. Incubate at 37 °C, 5% CO2 for 7 days.
    11. Centrifuge lymphocyte cell suspension 5 min 1,200 rpm (300 x g).
    12. Discard supernatant and wash cell pellet with 10 ml HBSS. Count viable cell number using 0.4% Trypan blue solution, a hemocytometer chamber and an optic microscope
    13. Centrifuge cell suspension 5 min 1,200 rpm (300 x g).
    14. Discard supernatant and resuspend 20 million lymphocytes in 1 ml diluent C of PKH26 Red Fluorescent Cell Linker Kit.
    15. Dilute 4 μl PKH26 dye in 1 ml diluent C (4 μM).
    16. Mix dye and cells (PKH26 at 2 μM final), invert the tube gently and incubate for 5 min RT.
    17. Add 2 ml FBS. Mix well. Incubate 1 min RT.
    18. Add 16 ml complete media. Mix well by inversion. Centrifuge cell suspension 10 min 1,200 rpm (300 x g).
    19. Discard supernatant and resuspend cell pellet in 10 ml complete media. Centrifuge cell suspension 10 min 1,200 rpm (300 x g).
    20. Repeat step 19 twice for a total of 3 washes.
    21. Count viable cell number using 0.4% Trypan blue solution, a hemocytometer chamber and an optic microscope.
    22. Resuspend PKH26-labeled lymphocytes at 2 million/ml (up to 50 million in 25 ml) in RPMI1640 media without FBS in a T25 vented tissue culture flask.
    23. Induce apoptosis by exposing lymphocytes to γ-irradiation (10 Gy).
    24. Incubate lymphocytes overnight 5% CO2, 37 °C in either complete media (for early apoptotic cells) RPMI without serum for late apoptotic cells at 2 million/ml.

  2. Isolation and culture of monocytes
    1. After recovering monocytes from elutriation chamber, wash in HBSS, count, and resuspend at 0.5 million/ml in complete media (Day 0).
    2. Add 10 ml per 100 mm petri dish.
    3. Add recombinant human M-CSF to a final concentration of 25 ng/ml.
    4. Place at 37 °C, 5% CO2.
    5. After 3-4 days, add 5 ml fresh complete media (containing 25 ng/ml M-CSF) per plate.
    6. On day 6-8, discard media from plates and wash adherent cells twice with 5 ml HBSS.
    7. Discard last HBSS wash and add 5 ml CellStripper to the plates, incubate 20-30 min RT.
    8. Pipet up and down to detach the cells and transfer to 50 ml conical tube containing 25 ml prewarmed complete media (one tube = 5 plates, final volume 50 ml).
    9. Centrifuge cell suspension 1,200 rpm (300 x g), 5 min RT.
    10. Wash cell pellet twice with 10 ml HBSS, centrifuge cell suspension 5 min 1,200 rpm (300 x g).
    11. Count viable cell number using 0.4% Trypan blue solution, a hemocytometer chamber and an optic microscope.
    12. Plate human monocyte derived macrophages (HMDM) at 0.25 million/ml in complete media, 500 cells/mm2. For immunocytochemistry (ICC), plate cells in 24-well plates containing 12 mm coverslips (0.5 ml).
    13. Incubate at 37 °C, 5% CO2 overnight.
  3. C1q binding to apoptotic lymphocytes
    1. Transfer apoptotic lymphocytes to conical tube. Assess apoptosis by flow cytometry using the apoptosis detection kit from Biovision.
    2. Centrifuge cell suspension 5 min 1,200 rpm (300 x g).
    3. Discard supernatant and carefully resuspend lymphocytes in 10 ml prewarmed HBSS. Count ALL cells (viable and permeable) using 0.4% Trypan blue solution, a hemocytometer chamber and an optic microscope.
    4. Centrifuge cell suspension 5 min, 1,200 rpm (300 x g).
    5. Resuspend cell pellet at 5 x 106 cells/ml in HBSS/1%HSA in sterile 12 x 75 mm round bottom tube.
    6. Depending on the number of C1q coated apoptotic cells desired, add human purified C1q to a final concentration of 150 μg/ml. Pipet gently up and down or invert to mix.
    7. Incubate for 1 h at 37 °C, gently shake tubes every 15 min.
    8. Add 2 ml HBSS per tube and centrifuge cell suspension 5 min, 1,200 rpm (300 x g).
    9. Discard supernatant, add 2 ml HBSS per tube and centrifuge cell suspension 5 min, 1,200 rpm (300 x g).
    10. Resuspend cell pellet at desired concentration in phagocytosis buffer for uptake assay. Set aside 2 x 105 apoptotic lymphocytes +/- C1q to assess C1q binding efficiency as described below.

  4. C1q binding efficiency
    1. Resuspend 2 x 105 apoptotic lymphocytes +/- C1q in 100 μl FACS buffer.
    2. Add 1-2 μl murine anti-human C1q and incubate for 30 min on ice.
    3. Add 2 ml FACS buffer, centrifuge cell suspension 5 min 1,200 rpm (300 x g), 4 °C.
    4. Discard supernatant and resuspend cell pellet in 100 μl FACS buffer.
    5. Add 1 μl FITC conjugated anti-mouse IgG and incubate for 30 min on ice in the dark.
    6. Add 2 ml FACS buffer, centrifuge cell suspension 5 min 1,200 rpm (300 x g), 4 °C.
    7. Discard supernatant and resuspend cell pellet in 200 μl FACS buffer.
    8. Analyze by flow cytometry to determine C1q binding efficiency to apoptotic lymphocytes. Percentage of apoptotic cells binding C1q should be greater than 50%.

  5. Uptake assay
    1. Recover human monocyte derived macrophages (HMDMs) plate from Section C. Discard media and wash adherent cells twice with HBSS.
    2. Recover apoptotic lymphocytes from step IV-10. Add apoptotic lymphocytes +/- C1q at a 5:1 ratio (for example 5 x 105 apoptotic lymphocytes for 1 x 105 HMDMs in phagocytosis buffer in a final total volume of 2 ml per a 6-well plate well).
    3. Centrifuge the plate 3 min at 700 rpm (100 x g).
    4. Incubate for 1 h at 37 °C.
    5. To assess uptake by flow cytometry:
      1. Discard media and wash adherent cells twice with HBSS.
      2. Add 0.5 ml 0.05% trypsin and incubate 2 min 37 °C. Pipet cells up and down and transfer into 12 x 75 mm tubes (microscopically check that all macrophages have been recovered).
      3. Centrifuge cells 5 min 1,200 rpm (300 x g), RT.
      4. Discard supernatant and resuspend cell pellet in 100 ml FACS buffer.
      5. Add 5 μl CD11b-FITC antibodies per 1 x 106 cells, incubate 45 min in the dark on ice.
      6. Add 2 ml FACS buffer, spin down 5 min 1,200 rpm (300 x g) 4 °C.
      7. Centrifuge cells 5 min 1,200 rpm (300 x g), RT.
      8. Discard supernatant and resuspend in 300 μl FACS buffer to read.
      9. Analyze by flow cytometry.
    6. To assess uptake by ICC (using 12 mm coverslips placed in a 24-well plate).
      1. After step E-5, discard media and wash adherent cells twice with 0.5 ml HBSS.
      2. Fix cells with 3.7% formaldehyde (300 μl/well), 10 min RT.
        Note: Do not use methanol or acetone as it can disrupt the PKH26 membrane labeling of apoptotic lymphocytes.
      3. Wash cells twice with PBS.
      4. Stain cells with 4U FITC-phalloidin per well diluted in 250 μl PBS for 20 min RT.
      5. Wash cells twice with PBS.
      6. Mount coverslips with a drop of prolong gold antifade reagent.
      7. Analyze by fluorescence or confocal microscopy.

Recipes

  1. Complete media
    RPMI1640 + L-Glutamine + HEPES (500 ml)
    50 ml (10%) heat-inactivated FBS
    5 ml (1%) Penicillin/Streptomycin
    5 ml (1%) 200 mM L-Glutamine
  2. Phagocytosis buffer
    RPMI1640 + L-Glutamine + HEPES
    25 mM HEPES
    5 mM MgCl2
  3. ACK buffer
    To 450 ml milliQ water add
    4.145 g Ammonium Chloride (anhydrous)
    0.5 g potassium bicarbonate
    18.6 mg disodium EDTA
    Adjust pH to 7.4
    Bring final volume to 500 ml with milliQ water
    Filter sterilize using stericup
  4. FACS buffer
    500 ml HBSS (no phenol red)
    0.2% NaN3
    0.2% BSA

Acknowledgments

Human peripheral blood lymphocytes and monocytes are isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986).

References

  1. Benoit, M. E., Clarke, E. V., Morgado, P., Fraser, D. A. and Tenner, A. J. (2012). Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells. J Immunol 188(11): 5682-5693.
  2. Bobak, D. A., Frank, M. M. and Tenner, A. J. (1986). Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP. J Immunol 136(12): 4604-4610.
  3.  Lionetti, F. J., Hunt, S. M., Valeri, C. R. (1980). Methods of Cell Separation. New York: Plenum Publishing Corp.

简介

为了表征在自体凋亡细胞摄取期间的巨噬细胞基因表达谱,我们开发了一种独特的,更生理的系统,使用通过非活化分离程序纯化的原代人单核细胞衍生的巨噬细胞(和在没有污染的血小板,如果激活可以释放刺激信号 )和自体淋巴细胞作为凋亡细胞的来源。 使用自体细胞作为凋亡靶而不是转化细胞系避免了来自HLA水平上的"非自身"结构的抗原刺激,而且还由于细胞系中固有的转化而导致的"改变的自身"信号。

关键字:凋亡细胞的清除, 自身免疫性疾病, 巨噬细胞, 人类, C1q

材料和试剂

  1. 人外周血
  2. RPMI1640 + L-谷氨酰胺+ HEPES(Life Technologies,目录号:22400-105)
  3. FBS(在56℃下灭活30分钟)(Hyclone定义的FBS,目录号:SH30070.03)
  4. 青霉素/链霉素(Life Technologies,目录号:15070-063)
  5. L-谷氨酰胺(200mM)(Life Technologies,目录号:25030-081)
  6. 0.4%台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  7. HBSS(Corning/Cellgro,目录号:MT-21-023-CV)
  8. PBS
  9. 25%人血清白蛋白(HSA)(Plasbumin -25)(Talecris Biotherapeutics,NDC编号:13533-684-20)
  10. 重组人IL-2(Peprotech,目录号:200-02)
  11. 重组人M-CSF(Peprotech,目录号:300-25)
  12. 牛血清白蛋白(BSA)(来自牛血清的白蛋白,冻干粉,≥96%)(Sigma-Aldrich,目录号:A2153-100G)
  13. C1q(Comptech,Texas,目录号:A099)
  14. 用于通用细胞膜标记的PKH26红色荧光细胞接头试剂盒(Sigma-Aldrich,目录号:PKH26GL)
  15. CellStripper离解试剂(Thermo Fisher Scientific,目录号:25-056-CI)
  16. 细胞凋亡检测试剂盒(BioVision,目录号:K101-100)
  17. 抗人C1q(Quidel,目录号:A201)
  18. FITC缀合的抗小鼠IgG(Jackson Immunoresearch,目录号:115-096-006)
  19. FcR阻断试剂人(Miltenyi,目录号:130-059-901)
  20. FITC缀合的抗人CD11b(Life Technologies,目录号:CD11B01)
  21. FITC小鼠IgG1同种型(Life Technologies,目录号:MG101)
  22. 荧光素鬼笔环肽(Life Technologies,目录号:F432)
  23. Stericup(Thermo Fisher Scientific,目录号:SCGVU11RE)
  24. 长寿黄金抗衰减试剂(Life Technologies,目录号:P36930)
  25. 氯化铵(无水)
  26. 碳酸氢钠
  27. EDTA二钠
  28. NaN 3
  29. 胰蛋白酶
  30. 完成媒体(见配方)
  31. 吞噬作用缓冲液(参见配方)
  32. ACK缓冲区(参见配方)
  33. FACS缓冲区(请参阅配方)

设备

  1. 50毫升锥形管(Thermo Fisher Scientific,目录号:339653) - 重要的单核细胞粘在其他管
  2. 12×75mm聚丙烯圆底无菌管(Thermo Fisher Scientific,目录号:14-956-1D)
  3. 12×75mm聚苯乙烯圆底管(Thermo Fisher Scientific,目录号:14-961-13)
  4. 组织培养板和通风瓶(任何尺寸)
  5. Eppendorf微量离心管
  6. 12mm盖玻片(Thermo Fisher Scientific,目录号:GG12),通过在70%乙醇中浸泡2×5分钟灭菌
  7. 非组织培养处理的100mm培养皿(Thermo Fisher Scientific,目录号:0875712) - 称为培养皿
  8. 组织培养罩
  9. 伽玛辐照器
  10. 5%CO 2,37℃加湿培养箱
  11. 离心机用于50ml锥形管和5ml底圆管
  12. 离心机带有用于板的旋转叶片转子(Sorvall,型号:RT7000或等同物)
  13. 光学和共聚焦荧光显微镜
  14. 血细胞计数器
  15. 流式细胞仪
  16. 自动移液器(全量程)
  17. 提示(全范围音量)
  18. 24孔板

程序


  1. 淋巴细胞分离,染色和凋亡诱导。
    1. 收集来自淘析的前两个50ml的流出物管,其含有淋巴细胞(单核细胞保留在淘析室中)。
    2. 以700rpm(100×g)离心淋巴细胞悬液10分钟,RT(室温),以除去大多数血小板。
    3. 弃去上清液和池细胞沉淀在10毫升ACK缓冲液(以删除残留的红细胞)。 孵育2-5分钟RT
    4. 加入20ml完全培养基。
    5. 以700rpm(100×g)离心细胞悬浮液10分钟,RT
    6. 弃去上清液并将细胞沉淀重悬在20ml HBSS中。 使用0.4%台盼蓝溶液,血细胞计数室和光学显微镜计数活细胞数。
    7. 以700rpm(100×g)离心细胞悬浮液10分钟,RT
    8. 在一个通风的组织培养瓶中,将完整培养基中的淋巴细胞以1百万/ml重悬
    9. 加入100U/ml重组人IL-2。
    10. 在37℃,5%CO 2孵育7天
    11. 离心淋巴细胞悬浮液5分钟1,200rpm(300×g)
    12. 弃去上清液并用10ml HBSS洗涤细胞沉淀。 使用0.4%台盼蓝溶液,血细胞计数室和光学显微镜计数活细胞数
    13. 离心细胞悬液5分钟1,200rpm(300×g)。
    14. 弃去上清液并在1ml稀释液C的PKH26红色荧光细胞接头试剂盒中重悬20万个淋巴细胞。
    15. 稀释4μlPKH26染料在1 ml稀释剂C(4μM)
    16. 混合染料和细胞(PKH26最终2μM),轻轻倒转试管,孵育5分钟RT。
    17. 加入2ml FBS。 混合好。 孵育1分钟RT。
    18. 加入16毫升完全培养基。 通过反转混合均匀。 将细胞悬浮液离心10分钟,1200rpm(300×g )
    19. 弃去上清液并将细胞沉淀重悬于10ml完全培养基中。 将细胞悬浮液离心10分钟,1200rpm(300×g )
    20. 重复步骤19两次,共进行3次洗涤。
    21. 使用0.4%台盼蓝溶液,血细胞计数室和光学显微镜计数活细胞数
    22. 在T25通气的组织培养瓶中,在不含FBS的RPMI1640培养基中以2百万/ml(在25ml中高达5000万)重悬PKH26标记的淋巴细胞。
    23. 通过将淋巴细胞暴露于γ-照射(10Gy)诱导凋亡。
    24. 在完全培养基(对于早期凋亡细胞)中,将淋巴细胞在37℃下孵育过夜5%CO 2,以2百万/ml的晚期凋亡细胞的无血清RPMI。

  2. 单核细胞的分离和培养
    1. 从淘洗室回收单核细胞后,在HBSS中洗涤,计数,并在完全培养基(第0天)中以0.5百万/ml重悬。
    2. 加入10毫升每100毫米培养皿。
    3. 加入重组人M-CSF至终浓度为25ng/ml
    4. 置于37℃,5%CO 2
    5. 3-4天后,每板加入5ml新鲜完全培养基(含25ng/ml M-CSF)。
    6. 在第6-8天,从培养皿中弃去培养基,用5ml HBSS洗涤贴壁细胞两次。
    7. 弃去最后一次HBSS洗涤,并向板中加入5 ml CellStripper,孵育20-30分钟RT
    8. 上下移动以分离细胞并转移至含有25ml预热的完全培养基(一个管= 5个板,终体积为50ml)的50ml锥形管中。
    9. 离心细胞悬浮液1200rpm(300×g),RT下5分钟
    10. 用10ml HBSS洗涤细胞沉淀两次,离心细胞悬液5分钟1,200rpm(300×g)。
    11. 使用0.4%台盼蓝溶液,血细胞计数室和光学显微镜计数活细胞数
    12. 将人单核细胞来源的巨噬细胞(HMDM)以0.25×10 6/ml在完全培养基中,500细胞/mm 2培养板上。 对于免疫细胞化学(ICC),平板细胞在含有12mm盖玻片(0.5ml)的24孔板中。
    13. 在37℃,5%CO 2中孵育过夜。
  3. C1q与凋亡淋巴细胞的结合
    1. 转移凋亡淋巴细胞到锥形管。 使用来自Biovision的凋亡检测试剂盒通过流式细胞术评估细胞凋亡
    2. 离心细胞悬浮液5分钟1,200rpm(300×g)
    3. 弃去上清液并小心地将淋巴细胞重悬在10ml预热的HBSS中。 使用0.4%台盼蓝溶液,血细胞计数室和光学显微镜计数ALL细胞(活的和可渗透的)。
    4. 将细胞悬浮液离心5分钟,1200rpm(300×g )
    5. 在无菌的12×75mm圆底管中,将细胞沉淀以5×10 6个细胞/ml悬浮于HBSS/1%HSA中。
    6. 根据所需的C1q包被的凋亡细胞的数量,将人纯化的C1q加至终浓度为150μg/ml。 轻轻地上下移动或倒转混合。
    7. 在37°C孵育1小时,每15分钟轻轻摇动管
    8. 每管加入2ml HBSS,并在1200rpm(300×g)下离心细胞悬浮液5分钟。
    9. 弃去上清液,每管加入2ml HBSS,并在1,200rpm(300×g)下离心细胞悬浮液5分钟。
    10. 在吞噬作用缓冲液中以所需浓度重悬细胞沉淀用于摄取测定。 放置2×10 5个凋亡淋巴细胞+/- C1q以评估如下所述的C1q结合效率。

  4. C1q结合效率
    1. 在100μlFACS缓冲液中重悬2×10 5个凋亡淋巴细胞+/- C1q。
    2. 加入1-2μl鼠抗人C1q,并在冰上孵育30分钟
    3. 加入2ml FACS缓冲液,离心细胞悬液5分钟1,200rpm(300×g),4℃。
    4. 弃去上清液并将细胞沉淀悬浮于100μlFACS缓冲液中。
    5. 加入1微升FITC结合的抗小鼠IgG,并在冰上在黑暗中孵育30分钟
    6. 加入2ml FACS缓冲液,在4℃下离心细胞悬浮液5分钟1,200rpm(300×g)。
    7. 弃去上清液并在200μlFACS缓冲液中重悬细胞沉淀
    8. 通过流式细胞术分析以确定C1q与凋亡淋巴细胞的结合效率。 结合C1q的凋亡细胞的百分比应大于50%。

  5. 摄取测定
    1. 回收来自部分C的人单核细胞衍生的巨噬细胞(HMDM)板。弃去培养基并用HBSS洗涤贴壁细胞两次。
    2. 恢复步骤IV-10的凋亡淋巴细胞。 在吞噬作用缓冲液中以5:1的比例加入凋亡淋巴细胞+/- C1q(例如5×10 5个凋亡淋巴细胞,1×10 5个HMDM),最终总体积 每6孔板孔2ml)。
    3. 以700rpm(100×g)将板离心3分钟。
    4. 在37℃下孵育1小时。
    5. 通过流式细胞术评估吸收:
      1. 弃去培养基,用HBSS洗涤贴壁细胞两次
      2. 加入0.5ml 0.05%胰蛋白酶并在37℃下孵育2分钟。 吸取细胞上下和转移到12×75毫米管(显微镜检查所有巨噬细胞已回收)。
      3. 离心细胞5分钟1,200rpm(300×g),室温
      4. 弃去上清液并将细胞沉淀重悬在100 ml FACS缓冲液中
      5. 每1×10 6个细胞加入5μlCD11b-FITC抗体,在冰上在黑暗中孵育45分钟。
      6. 加入2ml FACS缓冲液,在4℃下,以1200rpm(300×g)旋转5分钟。
      7. 离心细胞5分钟1,200rpm(300×g),室温
      8. 弃去上清液并重悬于300μlFACS缓冲液中以进行读数
      9. 通过流式细胞术分析
    6. 为了评估ICC的摄取(使用放置在24孔板中的12mm盖玻片)。
      1. 在步骤E-5之后,弃去培养基并用0.5ml HBSS洗涤贴壁细胞两次
      2. 用3.7%甲醛(300μl/孔)固定细胞,RT 10分钟。
        注意:不要使用甲醇或丙酮,因为它可能破坏凋亡淋巴细胞的PKH26膜标记。
      3. 用PBS洗涤细胞两次。
      4. 染色细胞用每孔4U FITC-鬼笔环肽在250μlPBS中稀释20分钟RT。
      5. 用PBS洗涤细胞两次。
      6. 安装盖玻片与一滴延长金抗衰减试剂。
      7. 通过荧光或共聚焦显微镜分析

食谱

  1. 填写媒体
    RPMI1640 + L-谷氨酰胺+ HEPES(500ml) 50ml(10%)热灭活的FBS 5ml(1%)青霉素/链霉素 5ml(1%)200mM L-谷氨酰胺
  2. 吞噬缓冲液
    RPMI1640 + L-谷氨酰胺+ HEPES
    25 mM HEPES
    5mM MgCl 2/
  3. ACK缓冲区
    向450ml milliQ水中加入
    4.145克氯化铵(无水)
    0.5克碳酸氢钠 18.6mg EDTA二钠
    将pH调节至7.4
    用milliQ水
    使最终体积达到500 ml 使用stericup过滤灭菌
  4. FACS缓冲区
    500ml HBSS(无酚红)
    0.2%NaN 3
    0.2%BSA

致谢

如先前所述(Bobak等人,1986),使用Lionetti等人(1980)的技术的修改通过逆流淘析分离人外周血淋巴细胞和单核细胞。 。

参考文献

  1. Benoit,ME,Clarke,EV,Morgado,P.,Fraser,DA和Tenner,AJ(2012)。人体吞噬细胞上C1q受体表达的表征:PDBu和fMLP的作用。 Immunol 136(12):4604-4610。
  2.   Lionetti,F.J.,Hunt,S.M.,Valeri,C.R。(1980)。细胞分离方法。纽约:Plenum出版公司。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Benoit, M. E., Clarke, E. V. and Tenner, A. J. (2013). C1q Binding to and Uptake of Apoptotic Lymphocytes by Human Monocyte-derived Macrophages. Bio-protocol 3(17): e877. DOI: 10.21769/BioProtoc.877.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。