Alexa Fluor-conjugated secondary antibodies, diluted 1 200 in blocking buffer, were added for 1 hour at room temperature.
After an incubation time of 10 min at room temperature, additional 400 μl of binding buffer were added for a final volume of 500 μl.
After three washes, anti-mouse or rabbit Europium-labeled secondary antibodies (1 μg/ml in assay buffer) were added for an additional hour.
Wells were washed twice with SM buffer and either 220 μl of a phage stock (10 PFU/ml) or 220 μl of SM buffer were added for test and control purposes, respectively.
The frozen whole blood with anticoagulant was thawed in a 37°C water bath and 4 volumes of red blood cell lysis buffer were added for lysing red blood cells.
Annexin V-FITC and PI (5 μl per 100 μl buffer) were added for 15 min at room temperature (RT) in darkness, and then analysed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) within 1 h.
After three washing steps with TBS, secondary antibodies (Alexa Fluor 350-conjugated rabbit anti-goat IgG [1 : 100] (Invitrogen, Karlsruhe, Germany) and Cy2-conjugated rabbit anti-mouse IgG [1 : 400] (Dianova, Hamburg, Germany)) diluted in staining buffer were added for 1 h.
Following three washes with TBS/T, horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) diluted in blocking buffer were added for 30 min at room temperature and washed three times with TBS/T.
After PBS-tween/PBS wash Cyanine 5 (Cy5) directly conjugated to tyramide (Perkin-Elmer, Waltham, MA, USA) at 1 50 dilution in amplification buffer was added for 10 min for target antibody detection.
