Erythrocytes Synthesis Lab Report

MATERIALS AND METHODS

Preparation of human erythrocytes

The 10 ml blood samples were withdrawn from three healthy volunteers with informed consent in heparin test tubes. After centrifuging at 1250 g for 10 minute, the plasma portion and buffy coat were removed and the remaining erythrocytes were washed 3 times using phosphate-buffered saline (PBS). The washed packed RBCs had a hematocrit of about 80%.

RBCs loading with FVIII

Stepwise hypotonic dialysis was used to load factor FVIII (Lyophilized powder from human plasma produced by Biotest, IBRF) in RBCs. Two ml of washed pack cells and 250 unit of FVIII were put into a dialysis bag (Sigma). Dialysis bag presoaked for 4 h in 50 ml PBS buffer containing 5 mM glucose. The bag was placed in a

Every 20 min, 30 ml of sterile distilled water was add¬ed into the outside buffer and repeated 5 times to make a dilutional factor of 2.5. The dialysis bag was transferred into 100 ml of isoos¬motic buffer; pH 7.45 and incubated at 37°C for 1 h. Generated pores of RBCs’ membrane loaded with FVIII reversed into normal in isotonic media. The isoosmotic buffer was contained: 0.036 mM KH2PO4/KOH, 0.04 mM MgCl2, 0.84 mM NaCl, 0.18 mM glucose, and 0.27 mM adenosine. The RBC-carriers washed 3–4 times in sterile phys-iological saline by centrifugation (1250 g, 10 min) to remove hemoglobin and unloaded factor

Plasma derived Factor VIII (Lyophilized powder from human plasma produced by Biotest, IBRF) was used as a positive control. The SDS-PAGE gels were stained by Coomassie Brilliant Blue R-250 (Merck, Germany). The SDS-PAGE gels were transferred to PVDF membrane (Roche, Germany). The membranes were incubated overnight at 4°C with 5% skim milk (Merck, Germany) in PBS as blocking solution, and washed in PBS. The membranes were over laid with mouse monoclonal anti human factor VIII antibody (abcam, U.K) and incubated for 2h at room temperature (RT) and washed with 0.2% Tween 20/PBS three times, 5 min each. Incubation was continued at RT for 1h with HRP-anti mouse secondary antibody according to the manufacture protocol (abcam, U.K). After a final wash separated proteins were visualized using ECL (Santa cruze, USA) substrate solution using chemiluminescense system (BioRad, USA).

Flow cytometry

Presence of FVIII in erythrocytes was detected by flowcytometry using specific monoclonal antibody (abcam, U.K) against FVIII. 5 μl of washed FVIII loaded erythrocytes diluted with 100 μl PBS incubated at 4 for 30 minutes with FVIII monoclonal antibody. Secondary antibody conjugated with FITC was added and incubated at 4 for 30 minutes again. Cells were analysed with partec