EEXTRACTION OF THE LIPOPOLYSACCHARIDE (LPS) FROM ESCHERICHIA COIL'S CELL WALL AND IT'S EVALUATION AS A MITOGEN FORPROLIFERATION B- LYMPHOCYTES BY MTT TEST

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TAVAKOL AFSHARI J.; SADEGHIAN A.; KHALILI M.

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Journal: Pathobiology Research Year:2004 | Volume:7 | Issue:1 Page(s): 39-48

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Title

EEXTRACTION OF THE LIPOPOLYSACCHARIDE (LPS) FROM ESCHERICHIA COIL'S CELL WALL AND IT'S EVALUATION AS A MITOGEN FORPROLIFERATION B- LYMPHOCYTES BY MTT TEST

Author(s)

TAVAKOL AFSHARI J. | SADEGHIAN A. | KHALILI M. | Issue Writer Certificate

Keywords

LIPOPOLYSACCHARIDE (LPS)Q4

EXTRACTIONQ4

PURIFICATIONQ4

MTT ASSAYQ4

Abstract

Purpose: Lipopolysaccharide (LPS) is a major constituent of gram negative bacteria cell wall. LPS consistes of three covalently linked components of (i) lipid A (endotoxine A), (ii) core (oligosaccharide) and (iii) the a-antigen polymer (polysaccharide). LPS preparations are used extensively for research in the elucidation of the LPS structure, metabolisme, toxicity and biosynthesis. The LPS is also used as a mitogen for B- lymphocytes proliferation. The purpose of this study was to evaluate the extracted LPS as a mitogen by the MTT ASSAY.Materials & methods: Escherichia coli was grown in the nutrient broth (NB) and after the harvesting, the bacteria were washed with ethannol, aceton or chloroform- methanol. The LPS was extracted by two different methods of Phenol- chloroform- petroleum ether (PCP) and Methanol-Chloroform (MC). In the MC method, the bacteria was sonicated and then the LPS was extracted. In the PCP method, Petroleum ether and chloroform were removed on rotary evaporator or in a high vacuum at a degree bellow 0°C and the precipitated LPS was washed two to three times with ether to remove any remaining phenol. We measured the B cell proliferation by using the MTT ASSAY. We also used the cell viability assay with the trypan blue dye as a qualitative assay. In the MTT9 assay different concentrations of extracted LPS and standard LPS (Sigma) were added to 96 well plates containing 2.5x104 cells/well. These cells were separated from the whole blood by ficoll and suspendd in a complete RPMI-1640 medium. We measured the 0.0 of the MTT dye at 570 nm and 620 nm respectively.Results: Our result showed that the cellular proliferation by the PCP-LPS was more than the MC-LPS, compared to the control (cells without LPS) and was similar to the standard LPS.Conclusion: In conclusion, the PCP-LPS has more mitogeneic activity compared to the MC-LPS.

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APA: Copy

TAVAKOL AFSHARI, J., SADEGHIAN, A., & KHALILI, M.. (2004). EEXTRACTION OF THE LIPOPOLYSACCHARIDE (LPS) FROM ESCHERICHIA COIL'S CELL WALL AND IT'S EVALUATION AS A MITOGEN FORPROLIFERATION B- LYMPHOCYTES BY MTT TEST. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), 7(1), 39-48. SID. https://sid.ir/paper/81227/en

Vancouver: Copy

TAVAKOL AFSHARI J., SADEGHIAN A., KHALILI M.. EEXTRACTION OF THE LIPOPOLYSACCHARIDE (LPS) FROM ESCHERICHIA COIL'S CELL WALL AND IT'S EVALUATION AS A MITOGEN FORPROLIFERATION B- LYMPHOCYTES BY MTT TEST. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES)[Internet]. 2004;7(1):39-48. Available from: https://sid.ir/paper/81227/en

IEEE: Copy

J. TAVAKOL AFSHARI, A. SADEGHIAN, and M. KHALILI, “EEXTRACTION OF THE LIPOPOLYSACCHARIDE (LPS) FROM ESCHERICHIA COIL'S CELL WALL AND IT'S EVALUATION AS A MITOGEN FORPROLIFERATION B- LYMPHOCYTES BY MTT TEST,” PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), vol. 7, no. 1, pp. 39–48, 2004, [Online]. Available: https://sid.ir/paper/81227/en

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