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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

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.

Keywords

Reduction of alama blue;Acute phase response;Proliferation of splenolcyte and PBMC;Broiler chicks

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