Measurements were taken using a logarithmic gain. Forward scatter (FSC, size) and side scatter (SSC, granularity) gates for RBCs were identified in control experiments JNK inhibitor using anti-glycophorin

A-PE labelled RBCs. The positive fluorescent gate was set using RBCs unlabelled with FITC-LA. For each measurement, 10,000 events were gated. PS positive cells were defined as all events falling within the preset FSC, SSC and positive fluorescent gates. RBCs were incubated in tonometers at 2% Hct for up to 60 min after which samples were fixed in the same solution as that used during incubation but with the addition of 0.3% glutaraldehyde. Control experiments AZD2281 price showed that this protocol was sufficient to maintain the RBC shape for several weeks. Sickling was assessed by light microscopy. Several hundred RBCs (typically 300–400) were counted using an Improved Neubauer haemocytometer (in five 1 mm × 1 mm squares, the central one and the four corners). Cell water content was measured by the wet weight − dry weight method [25]. In brief, RBCs were pelletted by centrifugation at 12,000 g for 10 min at 4 °C. The extruded pellet

was weighed immediately (to 0.01 mg) and again after drying for 18 h at 95 °C. Water content was expressed as ml water per g dry cell solids (ml/g dcs). Results are presented as single observations representative of at least 3 others, or as means ± S.E.M. of n observations. Where appropriate, comparisons were made using paired Student’s t tests, with p < 0.05 being considered significant. In the first series of experiments, the effect of o-vanillin (5 mM) was tested on sickling of RBCs from HbSS patients ( Fig. 1). In fully deoxygenated RBCs, there was only a small reduction in percentage sickling

(N.S.) in the presence of o-vanillin. At higher O2 tensions, nearer the P50 for O2 saturation of Hb, greater effects were observed, however, so that at an O2 tension of 15 mm Hg, sickling was inhibited by about 75% in the presence of o-vanillin ( Fig. 1). The effects of o-vanillin (5 mM) were then tested on the main cation before pathways which mediate solute loss and dehydration of RBCs from SCD patients, under fully oxygenated and fully deoxygenated conditions. Results are shown in Fig. 2 for RBCs from homozygous (HbSS) patients. In the presence of o-vanillin, KCC in oxygenated RBCs was substantially inhibited (by about 75%). Pre-treatment with o-vanillin for 30 min prior to flux measurement produced a slight increase in inhibition. In these RBCs, KCC activity was reduced by about half by deoxygenation and this residual oxygen-insensitive component of KCC was also sensitive to o-vanillin (inhibition of this component of KCC activity was 73 ± 13% without pre-treatment, means ± S.E.M., n = 5).