Fluostar optima

We isolated genomic DNA using the ammonium acetate precipitation method [66 ]. We checked the integrity of the DNA using a 0.8% agarose gel stained with SYBRsafe (Invitrogen). We measured DNA concentration using either a fluorometer (FLUOstar OPTIMA) or Nanodrop ND800 (Thermo Fisher Scientific).

The wells of the ELISA microtiter plates (Analyzer Ltd., Budapest, Hungary) were coated with D-dimer antigen, diluted in carbonate buffer (pH = 9.6). 100 μl aliquots were measured into the wells of the plates), and allowed to incubate at 4°C overnight. Then the plates were washed three times with washing-diluting PBS buffer, containing 0.05% Tween 20 (Diavet Ltd., Budapest, Hungary). Thereafter the cell culture supernatant, diluted 10-fold with washing-diluting buffer was added to each well, in a volume of 100 μl. RPMI media (Sigma-Aldrich Co.) supplemented with 10% fetal calf serum (Sigma-Aldrich Co.), was used as a negative control. After one hour of incubation at 37°C, the wells were washed three times. One-hundred μl of a peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson Immuno Research Labs Inc., West Grove, PA) diluted 2,500-fold with PBS-Tween-20 buffer, was added to wells. After 1 hour of incubation and washing procedure, 100 μl of tetramethylbenzidine substrate (TMB) (Diavet Ltd., Budapest, Hungary) was mesured into the wells, and allowed to stand for 6 minutes. The colour reaction was stopped with 50 μl of 4 N H2SO4 solution. Then the absorbance was mesured at 450 nm using a FLUOstar Optima (Thermo Fisher Scientific, Waltham, MA USA) microplate reader.