I have read several times that calcium and magnesium ions activate/prime neutrophils and should be avoided when isolating neutrophils from whole blood. Does this priniciple apply for tissue culture - I will be co-culturing neutrophils with cancer cell lines.
Sincere thanks
I work with cells differentiated into neutrophils and I grow them in complete RPMI media (which has some calcium nitrate and magnesium sulfate). They don't present any activation.
When working with neutrophils isolated from blood one should use HBSS without Ca and Mg, that's right.
I used the following (as you can see with both Magnesium and calcium) buffer successfully with PMN when loading the cells with calcein and after that culturing them for a short period of time in RPMI + FBS:
Stock buffer
20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM KH2 PO4
Just before use supplement with:
5 mM glucose, 1.0 mM CaCl2, and 0.5% (w/v) HSA.
Normal PMN have a short lifespan and, in the presence of calcium and magnesium, isolated PMN undergo degranulation quite readily. Degranulation results in PMN death and often other cells nearby are damaged. If you intend to coculture for longer than two or three hours at 37°, then degranulation will be very likely with divalent cations present.
Thank you Robert, this was exactly my concern. Sincere thanks for your comment.
Yi, PMN isolated from whole blood are very sticky cells. There are a few things you can do to prevent them from adhering. 1. You can keep them in buffer lacking calcium and magnesium and containing EDTA. 2. You can keep them chilled, at 4 to 15° C. 3. You can keep them in tubes or culture vessels that have very low electrostatic charge such as bacteriological grade plastic or Teflon or siliconized glass. Non-siliconized glass, tissue culture grade plastic, and even some bacteriological grade plastic have high electrostatic charge. As soon as you allow the PMN to warm up to 37°C in the presence of divalent cations, they will adhere to surfaces or to each other forming clumps. In the presence of 1% BSA or 10% FBS, the degree of adhesion will be reduced, but not prevented.
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