B19 infectivity and expression of P antigen. Each cell line (2 × 10⁶) was inoculated with B19 (1 × 10¹¹ copies of B19 DNA) for 30 minutes at 4°C and washed with PBS, pH 7.2, 3 times. Half of the cells in each group were used for evaluation of B19 adsorption (left column in panel A), and remaining cells in 3 mL RPMI containing 10% FBS were further incubated at 37°C for 48 hours to measure B19 DNA replication (right column in panel A) or to detect B19 protein (B). (A) B19 binding and replication of B19 in various cell lines. B19-infected cells were quantified for B19 DNA as described in “Materials and methods.” The left column (▨) is regarded as B19 adsorption, and the right column (▪) as B19 replication. The scale for B19 DNA is shown in logarithm. (B) Detection of B19 protein in B19-infected cells. After a 48-hour incubation with B19, the cells were washed 3 times with PBS and they were fixed with 4% paraformaldehyde followed by permeabilization with SAP buffer (0.1% saponin, 0.05% NaN₃ in Hanks balanced salt solution). Then, cells were incubated with PAR3 at a concentration of 5 μg/mL on ice for 30 minutes, followed by an incubation with FITC-conjugated goat anti–mouse IgG. The expression of B19 protein in cytoplasm was analyzed by flow cytometry with PAR3 (line) or isotype-matched antibody 1F5 (shadow; left panel), or by immunofluorescence (IF) staining with PAR3 (right panel). Two types of positive patterns were observed in flow cytometry: dull positive (DP) pattern in KU812Ep6, U937, H9, and ACHN; bright positive (BP) pattern in KU812Ep6. (C) Flow cytometry analysis of P antigen expression on the cell surface. Indicated cells were incubated with antigloboside antibody, GL4, followed by PE-labeled anti–rabbit IgG. Shadow represents staining using rabbit IgG as a negative control.