ABSTRACT

Background: For over 40 years the differential centrifugation and centrifugation by density gradient have been used for the isolation of subcellular organelles. However, these techniques do not always are preserved the biological properties of the organelles therefore is necessary to make technical changes to isolate organelles conserving its biological properties.
Methodology: We obtained derived monocytes to macrophages from 180 mL peripheral blood of healthy volunteers. Macrophages infected with Mycobacterium tuberculosis, homogenization mechanically and centrifuge using Percoll gradient to 27%. Subsequently, the homogenized was fractioned manually according to their density in 30 fractions.
Results: The organelles were separated according to their density. The plasma membrane with a density of 1,040 g/mL was localized in 10-14 fractions. Phagosomes and phagolysosomes with a density of 1,109 g/mL were found in 22-29 fractions. Characterization of subcellular organelles was performed by fluorometric methods for identifying specific, biotin coupling (plasma membrane), fractions containing the identification of Mycobacterium tuberculosis and fagolisosomal enzyme β-hexosaminidase.
Conclusions: The technique of ultracentrifugation in combination with enzymatic techniques useful to isolate and characterize phagosomes, phagolysosomes and plasma membrane. These organelles are involved in the processing and presentation of antigens and their isolation is crucial to study its role in the immune response.

KEYWORDS

Organelles, subcellular fractionation, ultracentrifugation, gradient of Percoll.

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