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Medium for neutrophils?

Medium for neutrophils? - Immunology and Host-Pathogen Interactions

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Old 08-31-2011, 02:41 AM
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Question Medium for neutrophils?



Hi everyone,

I am enriching neutrophils from peripheral blood and performing various assays. I am looking at protocols and it seems some recommend Ca and Mg free, others only Mg free and some also explicitly recomment Ca and Mg in the medium used.

Does anyone know why that is? In which circumstances would you need or avoid Ca and Mg?

Thanks for your help
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Old 09-02-2013, 01:11 PM
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Default Re: Medium for neutrophils?

Conditions with or without presence of alkaline earth metal (Ca2+, Mg2+) in the study of human neutrophils
For neutrophils isolation, assays for chronic granulomatous disease use buffer solution without Ca2+, Mg2+, and phenol red (e.g. Dulbecco’s modified Eagle’s medium [DMEM], Dulbecco’s phosphate-buffered saline [DPBS], 10X Hank’s balanced salt solution [HBSS].
For “Ester Loading” procedure for neutrophils is crucial that the loading medium contain Ca2+ (e.g., 1-2 mM) because as the Ca2+ chelator is generated within the cytosol, it will bind Ca2+ (the extent depending on its kd). Without additional extracellular Ca2+ to replace this, Ca2+ will be displaced from Ca2+ stores within the cell (or worse, not replaced at all).
Neutrophil Polarization, Chemotaxis and Phagosome formation use EGM-2MV for primary neutrophils and Gey’s medium for HL-60 cells : 138 mM NaCl, 6 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 20 mM HEPES, 1 mM CaCl2, 1 mM MgSO4 and 0.5% HAS.
Phagocytosis of C3bi-coated yeast particles by adherent neutrophils does not result in elevations in intracellular Ca2+; in contrast, the phagocytosis of immunoglobulin-coated particles does activate changes in Ca2+. Spreading can occur in the absence of extracellular Ca2+, but adherence-associated actin polymerisation requires the presence of extracellular Ca2+. The role of intracellular Ca2+ in degranulation is more clearly established. Changes in intracellular Ca2+ levels can be achieved by addition of different concentrations of Ca2+ ionophore, by varying the concentration of Ca2+ in the extracellular medium or by manipulation of intracellular Ca2+ levels using intracellular buffers. These approaches have shown that degranulation does not occur at intracellular concentrations <200 nM, whereas degranulation of specific and gelatinase-containing granules occurs at 610-650 nM Ca2+ and 2.5 ÁM Ca2+ is required for maximal release of azurophilic granules. However, these experiments were performed in populations of neutrophils in suspension, and it may be that these values are different for individual or adhered cells. Localised Ca2+ changes within the cell may also be involved in direction movement.
The microbicidal activity is quite dependent upon the ionic strength of the incubation medium, decreasing as the ionic strength or serum concentrations are increased. Its activity is optimal at around neutral pH but is inhibited by 5-10 mM Mg2+ and >0.1 M NaCl.
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