Within 6 h of collection, the red cell pellet was washed in sterile phosphate-buffered saline (PBS) and the buffy coat was removed. The packed cell volume was aliquoted into several vials and cryopreserved in glycerolyte (Baxter, Deerfield, IL, USA), as described previously . This method of storage is effective in preserving
the level of red cell CR1 . Upon thawing, the red cell pellet was washed twice and stored in Alsever’s solution (114 mM dextrose, 27 mM sodium citrate, 71 mM sodium chloride, pH 6·1) at 4°C, usually within the same day. When repeat assays were required, additional aliquots were thawed. In preliminary experiments we observed no difference in the level of CR1 between fresh and thawed frozen samples. Red www.selleckchem.com/products/jq1.html cell CR1 was measured using indirect fluorescent staining and flow cytometry. All procedures were as described previously . The IC was prepared as described previously . Rabbit anti-bovine serum albumin (BSA) and BSA (Sigma-Aldrich, St Louis, MO, USA) were made endotoxin-free by filtration through a polymyxin B column (Thermo Fisher Scientific, Inc., Waltham, MA, USA). In brief, 50 µl of 49 mg/ml rabbit anti-BSA and 3 µl of 5 mg/ml BSA were added to 950 µl BIBW2992 order of RPMI-1640 (Sigma-Aldrich).This was the point of equivalence for the
antigen–antibody reaction, as determined by turbidometric assay. After 1 h incubation at 37°C, the IC was kept at 4°C overnight. The formed IC was then centrifuged at 7800 g for 10 min at 4°C and the supernatant discarded. The insoluble
IC was washed three times by resuspending in sterile PBS. The protein concentration was determined by ultraviolet (UV) spectrophotometry of an aliquot solubilized in NaOH. The concentration of IC was adjusted to 700 µg/ml and the stock was stored at −70°C in 100 µl aliquots in endotoxin-free polypropylene tubes. The IC used for IC binding capacity assays was prepared as described above, Gefitinib datasheet except for the use of fluorescein isothiocyanate (FITC)-labelled BSA (Accurate Chemical Corp., Westbury, NY, USA). The IC binding capacity was measured as described previously . In brief, the anti-BSA : BSA-FITC IC was incubated with AB+ serum for 30 min at 37°C for opsonization. IC preparation to be used as unopsonized IC had 100 mM EDTA included in the cocktail. Opsonized and unopsonized ICs were added separately to wells containing 1 × 107 erythrocytes. The plate was covered with aluminium foil and incubated at 37°C for 30 min. The erythrocytes were washed thrice with ice-cold plain RPMI-1640. After aspiration of the supernatant, the erythrocytes were resuspended in 1% paraformaldehyde in PBS and stored at 4°C in the dark until flow cytometry performed within 24 h. A single healthy human immunodeficiency virus (HIV)-negative African adult was the source of macrophages for our experiments. Venous blood was drawn into heparinized vacutainers (Becton-Dickinson, San Diego, CA, USA).