Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
권호 표지
DOI QR코드

DOI QR Code

Proliferation Assay of Splenocyte and PBMC by the Evaluation of Alamar Blue Dye Reduction Value in Broiler Chicks

Alamar Blue 색소의 환원량 평가에 의한 급성기 반응중 육계병아리의 비장세포와 PBMC 증식도 측정

  • Im, J.T. (Department of Animal Life Sciences, College of Animal Life Sciences, Konkuk University) ;
  • Park, I.K. (Department of Animal Life Sciences, College of Animal Life Sciences, Konkuk University) ;
  • Koh, T.S. (Department of Animal Life Sciences, College of Animal Life Sciences, Konkuk University)
  • 임진택 (건국대학교 동물생명과학대학 동물생명과학부) ;
  • 박인경 (건국대학교 동물생명과학대학 동물생명과학부) ;
  • 고태송 (건국대학교 동물생명과학대학 동물생명과학부)
  • Published : 2007.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

In this study, hatched male broiler chicks(Ross) were fed on a basal diet and LPS was administered via intraperitoneal injection three times every other day, on the 9th, 11th and 13th days of the experiment, and then PBMC and splenocytes were isolated on day 14. The degree of alama blue reduction was evaluated at 4, 24, 48, 96 and 120 h in the splenocytes, and at 4, 8, 12, 24 and 48 h for PBMC of incubation after the addition of alama blue solution to the media. The cell numbers used in this experiment were 103, 104 and 105 cells per well, and the con A levels were 0.0, 1.0, 5.0, and 10.0 ㎍ per ml of medium. 1. The degree of alama blue reduction was found to increase in a linear fashion with increasing incubation time and cell numbers, for both splenocytes and PBMC. 2. During acute phase response, the degree to which alama blue was reduced was significantly elevated (p<0.05) at an incubation time of 24 hr for the splenocytes, 4 hr for PBMC, and a cell number of 105 cells per well, respectively. 3. The raised reduction of alama blue to control was linear with Con A levels in medium, and higher reduction in Con A 10.0 ㎍ relative to 1.0 or 5.0 ㎍ in ml medium was shown 4. The medium with autologous serum evidenced a significantly (p<0.05) higher reduction of alama blue relative to FBS. 5. Splenocytes and PBMC from the LPS-injected birds evidenced significantly higher levels of alama blue reduction regardless of incubation time, number of cells, level of Con A added, or serum type, as compared with what was observed in normal birds. The results indicated that the assay conditions for proliferative activity using the alama blue method in birds in which the acute phase response had been activated via intraperitoneal LPS injection requires 4 hrs of incubation for PBMC, 24 hrs of incubation for splenocytes, and 10㎍ of Con A per ml of medium.

급성기 반응중인 가금의 비장세포와 PBMC 증식도를 alamar blue(alamar blue) 환원량으로 측정하기 위한 조건들을 조사하였다. 갓 부화한 육계병아리(Ross 종) 수컷에 기초사료를 급여하고, 9, 11 및 13일령에 각각 복강내 LPS를 주입으로 급성기 반응중인 14일령 육계병아리의 혈액에서 PBMC와 비장에서 비장세포를 분리하였다. alamar blue 환원량은 비장세포에서는 4, 24, 48, 96 및 120시간, PBMC는 4, 8, 12, 24 및 48시간에 그리고 웰당 103, 104 및 105개로 분배하고, 배양액 ml 당 0.0, 1.0, 5.0 및 10.0㎍의 Con A를 첨가하며, 배양액 중 2.5%의 자가혈청과 FBS를 첨가하여 측정하였다. 1.alamar blue 환원량은 배양시간의 경과(R2=0.825~1.000)에 따라 그리고, 세포수(R2= 0.999)의 증가에 따라 직선적으로 증가하였다.2. 급성기 반응중인 병아리에서, alamar blue 환원량은 대조구에 비해 비장세포는 24시간이후, PBMC는 4시간이후부터 105개의 세포접종시 유의하게(p<0.05) 높았다.3.급성기 반응중인 병아리의 비장세포와 PBMC에서 alamar blue 환원량은 Con A 농도에 관계없이 대조구에 비해 높았으며, 배양액 ml 당 Con A 1.0 또는 5.0 ㎍ 첨가에 비해 10.0㎍ 첨가시 유의하게(p<0.05) 높았다.4. 그리고, 배양액중 2.5%의 자가혈청은 급성기 반응과 관계없이 FBS에 비해 alamar blue 환원량을 유의하게 높였다. 본 연구는 alamar blue 환원량 평가에 의한 급성기 반응중인 가금의 비장세포와 PBMC의 증식도 측정에는 alamar blue 첨가 후 PBMC는 4시간, 비장세포는 24시간 배양, 세포수는 웰 당 105개, 그리고, Con A는 배양액 ml 당 10.0 ㎍이 적당하다는 것을 나타낸다.

Keywords

References

  1. Abbas, A. L., Lichtman, A. H. and Pober, J. S. 2000. Cellular and Molecular Immunology. 4th edition. WB Saunders, Philadelphia, PA
  2. Ansar Ahmed, S., Gorgal, R. M. Jr. and Walsh, J. E. 1994. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [$^3H$]thymidine incorporation assay. J. Immunol. Methods 170:211-224 https://doi.org/10.1016/0022-1759(94)90396-4
  3. Ansar Ahmed, S., Gogal, R. M. Jr., Larsen, C. T. and Pierson, F. W. 1996. A novel non- radioactive diagnostic test for evaluating cell- mediated function in the chicken. Proc. XX world Poult. Congr. 2:533
  4. Back, S. A., Khan, R., Gan, X., Rosenberg, P. A. and Volpe, J. J. 1999. A new alamar blue viability assay to rapidly quantify oligodendrocyte death. J. Neurosci. Methods 91: 47-54 https://doi.org/10.1016/S0165-0270(99)00062-X
  5. Begg, A. C. and Mooren, E. 1989. Rapid fluorescence-based assay for radiosensitivity chemosensitivity testing in mammalian cells in vitro. Cancer Res. 49: 565-569
  6. Bernabei, P. A., Santini, V., Silvestro, L., Pozzo, O. D., Bezzini, R., Viano, I., Gattei, V., Saccardi, R. and Ferrini, P. R. 1989. In vitro chemosensi- tivity testing of leukemic cells: development of a semiautomated colorimetric assay. Hematol. Oncol. 7:243-253 https://doi.org/10.1002/hon.2900070307
  7. Chaby, R. 1999. Strategies for the control of LPS-mediated pathophysiological disorders. DDT 4: 209-221 https://doi.org/10.1016/S1359-6446(99)01336-7
  8. Cochet, O., Teillaud, J. L. and Sautes, C. 1998. Immunological techniques made easy. Wiley co, UK, pp.: 7
  9. de Fries, R. and Mitsuhashi, M. 1995. Quantifi- cation of mitogen induced human lymphocyte pro- liferation: comparison of Alamar Blue assay to $^3H$-thymidine incorporation assay. J. Clin. Lab. Anal. 9:89-95 https://doi.org/10.1002/jcla.1860090203
  10. Denizot, F. and Lang, R. 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89:271-277 https://doi.org/10.1016/0022-1759(86)90368-6
  11. Erf, G. F. 2004. Cell-mediated immunity in poultry. Poult. Sci. 83:580-590 https://doi.org/10.1093/ps/83.4.580
  12. Freshney, R. I. 1994. Culture of animal cells, third edition. Wileyliss, England, chapter 10
  13. Gazzano-Santoro, H., Ralph, P., Ryskamp, T. C., Chen, A. B. and Mukku, V. R. 1997. A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody. J. Immunol. Methods 202: 163-171 https://doi.org/10.1016/S0022-1759(97)00002-1
  14. Gogal, R. M. Jr., Ansar Ahmed, S. and Larsen, C. T. 1997. Analysis of avian lymphocyte proliferation by a new, simple, non-radioactive assay. Avian Disease 41: 714-725 https://doi.org/10.2307/1592166
  15. Goodwin, C. J., Holt, S. J., Downes, S. and Marshall, N. J. 1995. Microculture tetrazolium salts XTT and MTS. J. Immunol. Methods 179: 95-103 https://doi.org/10.1016/0022-1759(94)00277-4
  16. Gloeckner, H., Jonuleit, T. and Lemke, H. D. 2001. Monitoring of cell viability and cell growth in hollow-fiber bioreactor by use of the dye Alamar blue. J. Immunol. Methods 252:131-138 https://doi.org/10.1016/S0022-1759(01)00347-7
  17. Lee, J. K., Kim, D. B., Kim, J. I. and Kim, P. Y. 2000. In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants. Toxicol. in Vitro 14:345- 349 https://doi.org/10.1016/S0887-2333(00)00028-X
  18. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  19. Nakayama, G. R., Caton, M. C., Nova, M. P. and Parandoosh, Z. 1997. Assessment of the Alamar blue assay for cellular growth and viability in vitro. J. Immunol. Methods 204:205-208 https://doi.org/10.1016/S0022-1759(97)00043-4
  20. Nociari, M. M., Shalev, A., Benias, P. and Russo, C. 1998. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J. Immunol. Methods. 213:157-167 https://doi.org/10.1016/S0022-1759(98)00028-3
  21. O'Brien, J., Wilson, I., Orton, T. and Pognaa, F. 2000. Investigation of the alamar blue fluorscent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267:5421-5426 https://doi.org/10.1046/j.1432-1327.2000.01606.x
  22. Poltrak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C. and Du, X. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088 https://doi.org/10.1126/science.282.5396.2085
  23. Porstmann, T., Ternynck, T. and Avrameas, S. 1985. Quantitation of 5-bromo-2-deoxyuridine incorpor- ation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response. J. Immunol. Methods 82:169-179 https://doi.org/10.1016/0022-1759(85)90236-4
  24. Ralph, P. 1976. Differential toxicity of con- canavalin A and PHA on lymphoid and hematopoietic cell lines. In: Bittiger H, Schnebli HP, eds. Concanavalin A as a tool. New York: John Wiley and Sons, pp: 613-622
  25. SAS Institute Inc. 1988. SAS user's Guide, statistics version 5 ed. SAS Institute Inc., NC, USA
  26. Sawabe, Y., Yamagishi, H., Yamagishi, N., Yamamura, Y. and Oka, T. 1996. In vitro chemosensitivity of human pancreatic cancer cell lines. Int. J. Pancreatol. 20:185-190 https://doi.org/10.1007/BF02803767
  27. Schlager, S. I. and Adams, A. C. 1983. Use of dyes and radioisotopic markers in cytotoxicity tests. Methods Enzymol. 93: 233-245 https://doi.org/10.1016/S0076-6879(83)93045-8
  28. Squatrito, R. C., Connor, J. P. and Buller, R. E. 1995. Comparison of a novel redox dye cell growth assay to the ATP bioluminescence assay. Gynecol. Oncol. 58:101-105 https://doi.org/10.1006/gyno.1995.1190
  29. Verdrengh, M. and Tarkowski, A. 1997. Role of neutrophils in experimental septicemia and septic arthritis induced by Staphylococcus aureus. Infect. Immun. 65:2517-2521
  30. Zhi-Jun, Y. Striranganthan, N., Vaught, S. K., Arastu, S. and Ansar Ahmed. 1997. A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies. J. Immunol. Methods 26:25-39 https://doi.org/10.1016/0022-1759(79)90038-3
  31. 고태송, 임진택, 박인경, 김재환. 2004. 급성기 반응중인 육계병아리의 생산성에 미치는 사료중 크릴밀의 영향. 동물자원과학회지, 46: 173-182
  32. 고태송, 임진택, 박인경, 이혜정, 최도열, 최준영, 이홍구, 최윤재. 2005. 급성기 반응을 활성화한 육계병아리에서 사료중 미역제품 수준이 단백질과 에너지 대사에 미치는 영향. 동물자원과학회지 47:379-390
  33. 박인경, 김재환, 임진택, 고태송. 2004. 사료중 크릴밀을 급여한 육계의 생산성과 SOD 활성에 미치는 급성기 반응의 영향, 동물자원과학회지. 46:183-192
  34. 이혜정, 박인경, 임진택, 최도열, 최준영, 최종배, 이홍구, 최윤재, 고태송. 2005. 미역의 급여수준이 선천성 면역반응이 활성화한 육계병아리의 혈액 항산화 균형에 미치는 영향. 동물자원과학회지 47:29-38
  35. 임진택, 김재환, 박인경, 고태송. 2003. 살모넬라 LPS를 주입한 육계병아리의 생산성과 질소밸런스 및 대사에너지 이용성에 미치는 사료중 크릴밀의 영향, 동물자원과학회지. 45:957-966