Interestingly, many publications since also show a variation in the order of these steps, with sedimentation preceding density gradient centrifugation. In the method originally devised by Boyum, centrifugation on a density gradient, a concoction of a polysucrose (Ficoll) to aggregate erythrocytes and sodium diatrizoate (Hypaque) to adjust density, acts as the first step to separate out the peripheral blood mononuclear cells (PBMC), followed by dextran sedimentation to purify the neutrophils from the erythrocytes.
There are variations in the separation media used but dextran-based sedimentation is typically used in conjunction with a Ficoll-Hypaque density gradient centrifugation step. There are advantages and disadvantages between the uses of these techniques, but centrifugation on Ficoll-Hypaque medium and erythrocyte sedimentation techniques are the most commonly used methods. There are several different methods for isolating neutrophils from human blood, including the rapid “1-step” method of centrifugation on high-density Ficoll-Hypaque (1.114 g/mL) medium, “2-step” erythrocyte sedimentation on polysucrose-based media coupled with centrifugation on low-density Ficoll-Hypaque (1.077 g/mL) media, and discontinuous Percoll density gradient centrifugation, as well as flow cytometric cell sorting and immunomagnetic bead separation. This is important as it may alter the responsiveness of neutrophils to agonists and hence undermine the experimental findings or testing for neutrophil function in diagnostic and research laboratories. Some form of blood manipulation is required to achieve isolation of neutrophils from other blood constituents, and the challenge remains to minimise artefactual neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies.
Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation.
The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations.
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