Welcome guest, Sign in

Home

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

ELISpots Assay to Measure the Number of Murine Plasma Cells Producing Anti-dsDNA Antibodies

Immunology > Antibody analysis > Antibody-antigen interaction
Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Vol 1, Iss 1, 1/5/2012, 6653 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.34

[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, Mouse, ELISpots, Autoimmune, Anti-dsDNA

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.


How to cite this protocol: 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; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register