Neutralizing titer is defined as the reciprocal of highest serum dilution required for neutralization of 100 TCID50 of the virus in 50% of the wells. In general, serum is diluted in a twofold dilution series across the plate, using at least two rows of wells per serum and a volume of 50 μl. Then 50 μl unit volume of virus suspension containing about 100 TCID50 is added. After incubation of serum-virus mixture, cells are added.

My question is, while calculating VN titre, do you consider dilution of the serum alone (say 1/2, 1/4, 1/8, etc..) or the final dilution of serum in serum-virus mixture (1/4, 1/8, 1/16, etc..)? Appreciate your thoughts.