These hydrolases are normally confined at high concentrations in cytoplasmic vesicles (granules) and only released upon cell activation. Detergents can easily free the proteases from the granules. It was shown that even the presence of one PMN per million RBCs is able to release enough proteolytic power to damage, if not fully inhibited, highly sensitive RBC proteins such as ankyrin

and protein 4.1.6 KU-60019 Another common situation that could give rise to artefactual results is the preparation of “ghosts” from RBCs by hypotonic haemolysis.17 If the RBCs are contaminated by PMNs and the buffers used are not effectively supplemented with anti-proteases, the RBC membrane proteins will almost certainly be damaged (Fig. 1B, C). The workaround to this problem is the filtration of the blood and the use of freshly prepared lysing buffers containing a working concentration of anti-proteases. Other factors that must be standardised to be able to compare the obtained data between different laboratories are the temperature, shear stress, medium content, especially traces of serum, and the condition of cells used in the experiments. Furthermore, recent studies emphasise the importance of co-factors and substrates of several receptors, which may contribute to the experimental outcome. Temperature-related artefacts include ion misbalance and the ensuing changes in cell volume and Ca2 +-dependent

processes. Temperature ABT-199 purchase sensitivity depends on the particular approach, but it can be severe, differing, e.g., between different types of ion transporters. The decrease in the activity of ion transporters with a decrease in temperature by 10° (Q10) is approximately

30-fold for the Reverse transcriptase Ca2 + pump,18 approximately 3-fold for the Na+/K+ pump19 and approximately 1.5–3-fold for most of the ion transporter systems.[20] and [21] Thus, temperature changes may have a pronounced effect on the intracellular Ca2 + levels and the Na+/K+ distribution. The temperature may not necessarily be fixed at 37 °C in particular experimental settings (e.g., controlling the temperature can be complicate for patch-clamp investigations). However, temperature as a factor has to be taken into account, and the potential side effects must be controlled. Serum and the multiple biologically active factors it contains, including albumin and factors bound to it, such as interleukins, prostaglandins, insulin and amino acids, can introduce artefacts. Depending on the experimental settings, investigations are conducted in serum-containing or serum-free media. Proteins introduced with serum have been shown to play an active role in regulating the activity of ion transporters in RBCs obtained from healthy and diseased subjects. Little is known about the serum components mediating the effects. It has been shown that lysophosphatidic acid (LPA) activates Ca2 + uptake by RBCs.