The cell suspension was again mixed by inversion, incubated for five minutes at room temperature, and cells were pelleted (3' 13000 rpm), washed 1 ?with PBST and 1 ?with PBS (containing Mg2+ and Ca2+, as indicated for PBST) and resuspended in PBS with 2 goat serum and 2 BSA. Blocking was performed for 30' on ice at which point the bacteria were pelleted and resuspended in 100 of the blocking so