Hi Everyone,
In a phagocytosis paper I have read, it has said that "Phagocytes and prey were centrifuged (3000 × g, 1 min, including 30 s of acceleration) together prior to incubation for phagocytosis" : https://www.jimmunol.org/content/206/1/214 under 'Phagocytosis assay' of methods. Indeed, they found this significantly improved the amount of phagocytosis. Does anyone know why this is the case?
Thanks very much.
Subjecting the phagocytes and prey to a high speed centrifugation may be either to enhance the association under different treatment conditions or may be to check the effect of centrifugation on association.
The second thought makes more sense. Otherwise, macrophages are great eaters and just a simple co-culture for less than 1 hr will do the job.
- Hi Fiona,
- While working with human phagocyte THP-1 cells in our Xenotox, Inc. lab (http://www.xenobiovir.com/), we found the answer to the same question you asked in very recent paper of Therese de Neergaard et al. in J. Immunol., 2021(please find beneath).
- They showed that phagocyte–prey persistent association analysis can be used for data normalization, and concluded that persistent association normalization results in more robust and sensitive analysis. Namely, they applied that in a straightforward analytical method, persistent association-based normalization, in which the multiplicity of prey (MOP) ratio needed to elicit half of the phagocytes to associate persistently (MOP50) was determined first. MOP50 was then applied to normalize for experimental factors, separately analyzing association and internalization. As we did, authors used reference human phagocyte THP-1 cells with different prey and opsonization conditions to compare the persistent association-based normalization method to standard ways of assessing phagocytosis and find it to perform better, exhibiting increased robustness, sensitivity, and reproducibility. The approach might be easily incorporated into most existing phagocytosis assays (like ours) and allowed for reproducible results with high sensitivity.
- High-Sensitivity Assessment of Phagocytosis by Persistent Association-Based Normalization. T. de Neergaard, M. Sundwall, S. Wrighton, P. Nordenfelt. J Immunol ( January 1, 2021), 206(1) 214-224; DOI: https://doi.org/10.4049/jimmunol.2000032
Good luck,
Ilya
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