###### Abstract  
The influence of magnetic fields on the physico-chemical properties of water and aqueous solutions is well known. We have previously shown that weak combined magnetic fields with a 60 uT static component and a 100 nT (at 12.6 Hz) variable component are able to activate neutrophils, both directly and indirectly, through water pre-incubated in these fields. The ability to influence the activity of neutrophils was retained in serial dilutions of water, but only when a mechanical effect (shaking) was applied at each dilution step. Here, we confirm that combined magnetic fields are required for the formation of the stimulatory activity of water on ROS production by neutrophils. For the first time, we determined the threshold values of a constant magnetic field (at least 350-550 nT) necessary to maintain this activity in a series of successive dilutions. Additionally, the biophysical properties of various dilutions appeared to be not identical. This confirms that the number of technological steps (successive dilutions with physical influence) is a key factor that determines the activity of highly diluted samples. magnetic field; water; neutrophils; chemiluminescence; mechanical effect +
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Purified water (MilliQ water) subjected to the same magnetic treatment and subsequently added to a concentrated balanced salt solution and then to a suspension of these cells as an isotonic solution exerted a similar and even more pronounced stimulating effect compared to intact water (stored under geomagnetic field (GMF) conditions). Moreover, magnetically pre-treated water, subjected to multiple serial dilutions with vigorous shaking at each dilution step, retained its neutrophil-stimulating properties. At the same time, performing serial dilutions of magnetically pre-treated water, but at a near-zero magnetic field (inside a permalloy shielding chamber with a residual field of \(\sim\)10 nT) led to a complete loss of the ability to activate neutrophils. The stimulating properties were also demonstrated for diluted water that was pre-incubated in an artificial CMF (comparable with the GMF) generated inside the magnetically shielded chamber [11]. All these results indicate the critical role of the magnetic environment in the generation and retention of this biological activity in water. An important aim of this work was to demonstrate the dual role of the magnetic field during the preparation of HDs. Firstly, the magnetic field \"sets\" (forms) the defined activity in an aqueous solution, and secondly, it \"maintains\" this activity in the process of serial dilutions. In this paper, we will focus mainly on the second aspect, as well as on the issue of whether different dilutions with a dilution level of the initial substance exceeding \(10^{24}\) are equivalent to each other. Thus, this work does not examine a chemical whose concentration would decrease during the dilution process. We studied water after magnetic field treatment, and we conditionally called the process of mixing it with untreated water \"dilution\". It can be assumed that when diluting chemicals, a similar physical mechanism is realized; however, this phenomenon is more convenient to study in a system where solvent and solute molecules are identical. Additionally, this approach, in comparison with the study of a specific substance, allows us to address more substantively changes in the properties of water and the possibility of transmitting its effects in a series of dilutions using a physical mechanism. The results of this study will contribute to a better understanding of this phenomenon and can be useful for further research into the physico-chemical and biological properties of HDs. From a biological point of view, neutrophil granulocytes are one of the main types of cells that provide the first-line defense against pathological microorganisms, mainly through the phagocytosis and production of antimicrobial agents, including ROS, which can be detected using physico-chemical methods.