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Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells
骨髓源树突细胞中溶酶体pH值的测定   

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Abstract

Endosomes embraces different set of compartments such as early endosomes, intermediate endosomes and late endosomes or lysosomes. They become acidic as they mature. This acidification is generated by the vacuolar membrane proton pump V-ATPase that is recruited in late endosomes. This protocol described the measurement of endosomal pH using dextran molecules labelled with pH sensitive and insensitive dyes.

Materials and Reagents

  1. CO2 independent medium (Invitrogen, catalog number: 18045054 )
  2. Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, catalog number: I3390 )
  3. 10% fetal bovine serum (FBS) (Hyclone/PAA, catalog number: sv143-03 )
  4. Penicillin-streptomycin (100 Units/ml, 100 μg/ml) (Sigma-Aldrich, catalog number: P11-010 )
  5. Glutamine (Sigma-Aldrich, catalog number: G75013 )
  6. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  7. Granulocyte-macrophage colony stimulating factor (GM-CSF) (Peprotech, catalog number: 315-03 )
  8. 40,000 MW Dextran fluorescein (10 mg/ml) (Molecular Probes)
  9. 40,000 MW Dextran Alexa 647 (10 mg/ml) (Molecular Probes)
  10. 5 mM EDTA (Invitrogen, catalog number: 15575-038 )
  11. 1% Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  12. Triton X-100 (Sigma-Aldrich, X-100) kept at room temperature
  13. 1x PBS (see Recipes)
  14. Conditioned complete medium (see Recipes)

Equipment

  1. Water bath (37 °C)
  2. Incubator (37 °C)
  3. The FACSCalibur flow cytometer (Becton Dickinson)
  4. Hemocytometer
  5. Centrifuge

Procedure

  1. Detach bone marrow-derived dendritic cells (BMDCs) with 1x PBS-5 mM EDTA (10 min at 37 °C).
  2. Wash the cells with 1x PBS twice by centrifugation at 367 x g for 10 min.
  3. Count cells. A total of 3 x 106 cells are required for pH measurement at different time points (kinetics of 10 min, 20 min, 30 min and 60 min for example). Additionally, 6 x 106 cells are needed to acquire the pH standard curve.
  4. For measurement of pH at different time points, resuspend the cells in 100 μl total volume of prewarmed conditioned complete medium containing 1 mg/ml of fluorescein- and 0.5 mg/ml Alexa-647-labeled 40,000 MW dextrans. Pulse cells in a water bath set at 37 °C for 10 min.
  5. Stop the reaction by adding a large volume (1 ml) of cold 1x PBS-1% BSA and wash cells extensively (6 times) with the same buffer to get rid of the non-internalised dextrans.
  6. After washing, resuspend the cells in prewarmed conditioned complete medium (1 ml) and incubate at 37 °C for different time points (chase). The pulse (10 min) corresponds to early endosomes (EE), the chase of 40 min corresponds to intermediate endosomes (IE) and the 110 min of chase corresponds to the lysosomes.
  7. Stop the reaction each time by immediately adding cold PBS and placing the tubes on ice.
  8. Rapidly, analyse the cells by FACS, via a FL1/FL4 gate selective for cells that have endocytosed both fluorescent probes and determine the ratio of the mean fluorescence intensity (MFI) emission between the two probes.
  9. For the pH standard curve, resuspend the cells in 200 μl total volume of prewarmed conditioned complete medium containing 1 mg/ml of fluorescein- and 0.5 mg/ml Alexa-647-labeled 40,000 MW dextrans and pulse the cells in a water bath set at 37 °C for 20 min. Repeat step 5 and split cells into nine 1.5 ml Eppendorf tubes for a pH measurement ranging from 4 to 8.
  10. Prepare several buffers that differ in pH by 0.5 units using prewarmed CO2 independent medium. Adjust the pH with citric acid or NaOH.
  11. Resuspend each cell pellet in a different pH solution supplemented with 0.001% of Triton X-100 to slightly permeabilise the cells and to give access to the external prefixed pH solution into the cell.
  12. Analyse immediately by FACS and determine the ratio of the mean fluorescence intensity (MFI) emission between the two fluorescent probes at each pH. Make the standard curve by plotting the different MFI ratio values that correspond to each pH and apply this formula to the MFI ratio values obtained before (steps 1-8, Figure 1).





    Figure 1. Kinetic of endo-lysosomal pH in BMDCs pulsed with a mixed of fluorescein- and Alexa-647-labeled 40,000 MW dextrans for 10 min and then chased for different times

Recipes

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    8 mM Na2HPO4
    1.46 mM KH2PO4
    Keep 1x PBS cold
  2. Conditioned complete medium
    IMDM supplemented with
    10% FBS
    100 Units/ml,100 μg/ml penicillin-streptomycin
    2 mM glutamine
    50 μM 2-mercaptoethanol
    10 ng/ml GM-CSF

Acknowledgments

This protocol is adapted from Savina et al. (2010); Maschalidi et al. (2012) and Sepulveda et al. (2009).

References

  1. Maschalidi, S., Hassler, S., Blanc, F., Sepulveda, F. E., Tohme, M., Chignard, M., van Endert, P., Si-Tahar, M., Descamps, D. and Manoury, B. (2012). Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS Pathog 8(8): e1002841.
  2. Savina, A., Vargas, P., Guermonprez, P., Lennon, A. M. and Amigorena, S. (2010). Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays. Methods Mol Biol 595: 383-402.
  3. Sepulveda, F. E., Maschalidi, S., Colisson, R., Heslop, L., Ghirelli, C., Sakka, E., Lennon-Dumenil, A. M., Amigorena, S., Cabanie, L. and Manoury, B. (2009). Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells. Immunity 31(5): 737-748.

简介

内体包含不同组的区室,例如早期内体,中间内体和晚期内体或溶酶体。 它们成熟时变成酸性。 这种酸化由在后期内体中招募的液泡膜质子泵V-ATP酶产生。 该协议描述了使用用pH敏感和不敏感染料标记的葡聚糖分子测量内体pH。

材料和试剂

  1. CO 2独立培养基(Invitrogen,目录号:18045054)
  2. Iscove's Modified Dulbecco's Medium(IMDM)(Sigma-Aldrich,目录号:I3390)
  3. 10%胎牛血清(FBS)(Hyclone/PAA,目录号:sv143-03)
  4. 青霉素 - 链霉素(100单位/ml,100μg/ml)(Sigma-Aldrich,目录号:P11-010)
  5. 谷氨酰胺(Sigma-Aldrich,目录号:G75013)
  6. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  7. 粒细胞 - 巨噬细胞集落刺激因子(GM-CSF)(Peprotech,目录号:315-03)
  8. 40,000MW葡聚糖荧光素(10mg/ml)(Molecular Probes)
  9. 40,000MW葡聚糖Alexa 647(10mg/ml)(Molecular Probes)
  10. 5mM EDTA(Invitrogen,目录号:15575-038)
  11. 1%牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  12. Triton X-100(Sigma-Aldrich,X-100)保持在室温下
  13. 1x PBS(请参阅配方)
  14. 条件完全培养基(见配方)

设备

  1. 水浴(37℃)
  2. 培养箱(37℃)
  3. FACSCalibur流式细胞仪(Becton Dickinson)
  4. 血细胞计数器
  5. 离心机

程序

  1. 用1x PBS-5mM EDTA(37℃下10分钟)分离骨髓来源的树突细胞(BMDC)。
  2. 用1×PBS洗涤细胞两次,在367×g离心10分钟
  3. 计数单元格。在不同时间点(例如,10分钟,20分钟,30分钟和60分钟的动力学)下,需要总共3×10 6个细胞用于pH测量。另外,需要6×10 6个细胞来获得pH标准曲线
  4. 对于在不同时间点的pH的测量,将细胞重悬在100μl总体积的含有1mg/ml荧光素和0.5mg/ml Alexa-647标记的40,000MW葡聚糖的预热条件完全培养基中。脉冲细胞在37℃水浴10分钟。
  5. 通过加入大体积(1ml)冷的1×PBS-1%BSA停止反应,并用相同的缓冲液广泛(6次)洗涤细胞以除去非内化的葡聚糖。
  6. 洗涤后,将细胞重悬在预热的条件完全培养基(1ml)中,并在37℃下孵育不同的时间点(追踪)。脉冲(10分钟)对应于早期核内体(EE),40分钟的追踪对应于中间体内体(IE),追踪的110分钟对应于溶酶体。
  7. 通过立即加入冷PBS并将管置于冰上,停止反应
  8. 快速,通过FACS,通过FL1/FL4门选择性细胞分析细胞,内吞了两个荧光探针,并确定两个探针之间的平均荧光强度(MFI)发射的比率。
  9. 对于pH标准曲线,将细胞重悬在200μl总体积的含有1mg/ml荧光素和0.5mg/ml Alexa-647标记的40,000MW葡聚糖的预热条件完全培养基中,并将细胞在设定为37℃20分钟。重复步骤5,将细胞分成9个1.5 ml Eppendorf管,pH范围为4至8.
  10. 使用预热的CO 2独立培养基制备几个pH不同0.5个缓冲液。用柠檬酸或NaOH调节pH
  11. 将每个细胞沉淀重悬在补充有0.001%Triton X-100的不同pH溶液中以轻微渗透细胞,并获得外部预混pH溶液到细胞中。
  12. 立即通过FACS分析并确定在每个pH下两个荧光探针之间的平均荧光强度(MFI)发射的比率。通过绘制对应于每个pH的不同MFI比值并将该公式应用于之前获得的MFI比值(步骤1-8,图1),制备标准曲线。





    图1.用荧光素和Alexa-647标记的40,000MW葡聚糖的混合物脉冲10分钟,然后追踪不同时间的BMDC中内溶酶体pH的动力学。

食谱

  1. 1x PBS
    137 mM NaCl 2.7 mM KCl
    8mM Na 2 HPO 4
    1.46mM KH 2 PO 4>/
    保持1x PBS冷/
  2. 条件完全培养基
    IMDM补充
    10%FBS
    100单位/ml,100μg/ml青霉素 - 链霉素 2mM谷氨酰胺 50μM2-巯基乙醇 10ng/ml GM-CSF

致谢

该协议改编自Savina等人(2010); Maschalidi等人(2012)和Sepulveda等人(2009)。

参考文献

  1. Maschalidi,S.,Hassler,S.,Blanc,F.,Sepulveda,F.E.,Tohme, Chignard,M.,van Endert,P.,Si-Tahar,M.,Descamps,D。和Manoury,  (2012)。 天门冬酰肽酶内肽酶通过TLR7激活来控制抗流感病毒免疫反应。 Pathog 8(8):e1002841。
  2. Savina,A.,Vargas,P.,Guermonprez,P.,Lennon,A.M。和Amigorena,S。(2010)。 使用基于荧光测定法的测定法测量树突状细胞吞噬体中的pH,ROS产生,成熟和降解。/a> Methods Mol Biol 595:383-402。
  3. Sepulveda,FE,Maschalidi,S.,Colisson,R.,Heslop,L.,Ghirelli,C.,Sakka,E.,Lennon-Dumenil,AM,Amigorena,S.,Cabanie,L.and Manoury, 2009)。 天冬酰胺肽链内切酶在树突状细胞内吞Toll样受体信号传递中的关键作用 < em> Immunity 31(5):737-748。
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Copyright: © 2013 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. Maschalidi, S. and Manoury, B. (2013). Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells. Bio-protocol 3(14): e819. DOI: 10.21769/BioProtoc.819.
  2. Maschalidi, S., Hassler, S., Blanc, F., Sepulveda, F. E., Tohme, M., Chignard, M., van Endert, P., Si-Tahar, M., Descamps, D. and Manoury, B. (2012). Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS Pathog 8(8): e1002841.
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