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ELISpots Assay to Measure the Number of Murine Plasma Cells Producing Anti-dsDNA Antibodies
酶联免疫斑点法(ELISpot)检测产抗dsDNA抗体的鼠源浆细胞数量   

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Abstract

Circulating anti-dsDNA antibody is a hallmark of SLE both in human patients and in many SLE mouse models. This protocol describes how to measure the frequency of plasma cells producing these antibodies in the spleen of lupus-prone mice. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: Lupus(系统性红斑狼疮), Mouse(小鼠), ELISpots(Elispots), Autoimmune(自身免疫性), Anti-dsDNA(抗dsDNA抗体)

Materials and Reagents

  1. Salmon sperm DNA (Life Technologies, Invitrogen™, catalog number: AM9680 )
  2. Dulbecco’s modification eagle medium (DMEM) (Life Technologies, Invitrogen™)
  3. Ammonium chloride (0.17 M, filtered, autoclaved) (pH 7.4)
  4. 10% DMEM: 10% FBS in DMEM
  5. Biotin conjugated anti-IgM or anti-IgG (Southern Biotech)
  6. Streptavidin alkaline phosphatase (Southern Biotech)
  7. 5-Bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich)
  8. 10x PBS-Tween 20 (see Recipes)
  9. Blocking solution (see Recipes)
  10. AMP buffer (see Recipes)

Equipment

  1. 45-μm syringe top filter (USA Scientific)
  2. Cell-culture incubator
  3. Clear 96-well Microtest polystyrene assay plate (BD Biosciences)
  4. Conical tubes (Corning)
  5. Dissecting microscope or an ELISpot reader

Procedure

  1. Make double stranded salmon sperm DNA by passage though a 45 μm filter.
  2. Add 100 μl/well of 100 μg/ml salmon sperm DNA to a 96-well Microtest assay plate.
  3. Wrap the plate with plastic wrap and incubate at 4 °C for overnight.
  4. Discard the coating antibody solution and wash the plate with 1x PBS-Tween 6 times.
  5. Dry the plate and add 100 μl of blocking solution per well to the plate.
  6. Incubate the plate at room temperature (RT) for 1.5 h.
  7. Discard the blocking solution and wash the plate with 1x PBS-Tween 5 times.
  8. Dry the plate and keep it at 4 °C for later use.
  9. Harvest the spleen and create single cell suspension by gently smashing spleen pieces with the frosted surface of a pair of microscope slides in 5 ml of DMEM.
  10. Transfer the cells into 50 ml conical tubes and spin down the cells at 300 RCF for 5 min at 4 °C.
  11. Discard the supernatant with aspiration without disturbing the pellet.
  12. Resuspend the cells with 5 ml of 0.17 M ammonium chloride and keep the cells on ice for 5 min.
  13. Add 15 ml DMEM to the cells and spin at 300 RCF for 5 min at 4 °C.
  14. Discard the supernatant and resuspend the cells with 20 ml of DMEM and count the cells.
  15. Resuspend 2 x 107 cells in 2 ml of 10% DMEM and make three fold serial dilution (a total of 8 dilutions) with 10% DMEM.
  16. Add 50 μl/well of the serial dilutions on the DNA coated plate and centrifuge at 300 RCF for 5 min at 4 °C.
  17. Incubate the cells at 30 °C for 2 h in a cell-culture incubator with 6% CO2.
  18. Add 50 μl/well of biotin-conjugated anti-IgM or anti-IgG (1:350 in 10% DMEM) to the cells.
  19. Centrifuge the cells at 300 RCF for 5 min at 4 °C and incubate the cells overnight a cell-culture incubator with 6% CO2.
  20. Discard the cells and wash the plates 10 times with 10x PBS-Tween 20.
  21. Dry the plates and add 50 μl of streptavidin alkaline phosphatase (1:1,000 in 1% BSA/PBS) to the plate.
  22. Incubate the plate at RT for 1 h and wash the plate 10 times with 10x PBS-Tween 20.
  23. Dry the plate and add 50 μl/well of 1 mg/ml BCIP in AMP buffer to develop the plate.
  24. When the spots are clearly visible under a dissecting microscope, stop the development by discarding the BCIP solution and rinsing the plate with tap water thoroughly.
  25. Spots can be counted using a dissecting microscope or using an ELISpot reader.

Recipes

  1. 10x PBS-Tween 20 [0.1 M PBS, 0.5% Tween 20 (pH 7.4)]
    Na2HPO4 (anhydrous) 
    10.9 g
    NaH2PO4 (anhydrous)
    3.2 g
    NaCl
    90 g
    Distilled water
    1,000 ml
    Mix to dissolve and adjust pH to 7.4 and then add 5 ml of Tween 20, store this solution at RT. Dilute 1:10 with distilled water before use and adjust pH if necessary.
  2. Blocking solution
    5% FBS and 3% BSA in PBS
  3. AMP buffer (pH 10.25)
    0.75 mM MgCl
    0.01% Triton-X
    9.58% 2-amino-methyl-1propanol

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Mihara, M., Tan, I., Chuzhin, Y., Reddy, B., Budhai, L., Holzer, A., Gu, Y. and Davidson, A. (2000). CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J Clin Invest 106(1): 91-101.

简介

循环抗dsDNA抗体是人类患者和许多SLE小鼠模型中SLE的标志。 该协议描述如何测量产生这些抗体的浆细胞在脾脏易发小鼠的脾脏的频率。 这个协议是在费恩斯坦医学研究所的Anne Davidson博士的实验室中开发或修改的。

关键字:系统性红斑狼疮, 小鼠, Elispots, 自身免疫性, 抗dsDNA抗体

材料和试剂

  1. 鲑鱼精子DNA(Life Technologies,Invitrogen TM,目录号:AM9680)
  2. Dulbecco改良Eagle培养基(DMEM)(Life Technologies,Invitrogen TM)
  3. 氯化铵(0.17M,过滤,高压灭菌)(pH 7.4)
  4. 10%DMEM:10%FBS的DMEM培养基中
  5. 生物素缀合的抗IgM或抗IgG(Southern Biotech)
  6. 链霉亲和素碱性磷酸酶(Southern Biotech)
  7. 5-溴-4-氯-3-吲哚基磷酸酯(Sigma-Aldrich)
  8. 10x PBS-Tween 20(参见配方)
  9. 阻止解决方案(参见配方)
  10. AMP缓冲区(参见配方)

设备

  1. 45-μm注射器顶部过滤器(USA Scientific)
  2. 细胞培养孵化器
  3. 清洁96孔微量聚苯乙烯测定板(BD Biosciences)
  4. 圆锥管(Corning)
  5. 解剖显微镜或ELISpot阅读器

程序

  1. 通过45微米过滤器制作双链鲑鱼精子DNA。
  2. 将100μl/孔的100μg/ml鲑鱼精子DNA加入96孔Microtest测定板。
  3. 用塑料包裹包装板,并在4℃孵育过夜
  4. 弃去涂层抗体溶液,并用1×PBS-Tween洗板6次
  5. 干燥平板,每孔加入100μl封闭溶液到平板上
  6. 在室温(RT)下孵育平板1.5小时
  7. 弃去封闭液,用1×PBS-Tween洗板5次
  8. 干燥板,并保持在4°C以备后用
  9. 收获脾脏,通过用5ml的DMEM中的一对显微镜载玻片的磨砂表面轻轻地捣碎脾脏片段来产生单细胞悬浮液。
  10. 转移细胞到50毫升锥形管,并在300 RCF下旋转5分钟在4℃的细胞。
  11. 弃去上清液,不要打扰沉淀。
  12. 用5ml 0.17M氯化铵重悬细胞,并将细胞在冰上保持5分钟
  13. 加入15毫升DMEM的细胞,并在300 RCF在4℃下旋转5分钟
  14. 弃去上清液并用20ml DMEM重悬细胞并计数细胞
  15. 在2ml 10%DMEM中重悬2×10 7个细胞,并用10%DMEM进行三倍系列稀释(共8次稀释)。
  16. 在DNA包被的板上加入50μl/孔的连续稀释液,并在4℃下以300RCF离心5分钟。
  17. 孵育细胞在30℃下在具有6%CO 2的细胞培养孵育器中2小时。
  18. 加入50μl/孔的生物素结合的抗IgM或抗IgG(在10%DMEM中1:350)到细胞。
  19. 在4℃下以300RCF离心细胞5分钟,并将细胞在具有6%CO 2的细胞培养孵育器中孵育过夜。
  20. 弃去细胞,用10x PBS-Tween 20洗板10次
  21. 干燥平板并加入50μl链霉亲和素碱性磷酸酶(1:1,000在1%BSA/PBS中)到板中。
  22. 在RT孵育板1小时,并用10x PBS-Tween 20洗板10次
  23. 干燥板,加入50μl/孔的1mg/ml BCIP在AMP缓冲液中以显色板。
  24. 当斑点在解剖显微镜下清晰可见时,通过丢弃BCIP溶液并用自来水彻底冲洗板来停止显影。
  25. 可以使用解剖显微镜或使用ELISpot读数器计数斑点。

食谱

  1. 10x PBS-Tween 20 [0.1M PBS,0.5%Tween 20(pH 7.4)]
    Na 2 HPO 4(无水)
    10.9克
    NaH 2 PO 4(无水)
    3.2克
    NaCl
    90克
    蒸馏水
    1000 ml
    混合溶解并调节pH至7.4,然后加入5ml吐温20,将该溶液在室温下储存。 在使用前用蒸馏水稀释1:10,必要时调节pH值
  2. 封锁解决方案
    5%FBS和3%BSA的PBS溶液中
  3. AMP缓冲液(pH 10.25)
    0.75mM MgCl 0.01%Triton-X / 9.58%2-氨基 - 甲基-1-丙醇

致谢

该方案在Anne Davidson博士在Feinstein Institute for Medical Research,NY,USA的实验室中开发或修改。 这项工作得到NY SLE基金会(RB),Rheuminations,NIH AI082037和AR 049938-01,NIH(PO1 AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核)的赠予的支持。

参考文献

  1. Mihara,M.,Tan,I.,Chuzhin,Y.,Reddy,B.,Budhai,L.,Holzer,A.,Gu,Y.and Davidson,A。(2000)。 CTLA4Ig抑制小鼠系统性红斑狼疮中的T细胞依赖性B细胞成熟。 em Clin J Clin Invest 106(1):91-101。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Z. (2011). ELISpots Assay to Measure the Number of Murine Plasma Cells Producing Anti-dsDNA Antibodies. Bio-protocol 1(1): e34. DOI: 10.21769/BioProtoc.34.
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