Raji cells were washed with RPMI-1640 medium containing 10% FBS. After cell counting, cells were resuspended in RPMI-1640 medium containing 10% FBS diluted to 5 × 10 4 cells/mL. After centrifuging the suspension and removal of supernatant, 10% FBS RPMI-1640 medium containing calcein-AM (Dojindo) was added to be 1 mL (final concentration: 0.5 μM) and incubate at 37 °C under 5% CO2 for 30 min. Then, the suspension was centrifuged to remove the supernatant and 10% FBS RPMI-1640 medium containing probenecid (Fujifilm Wako) was added (1 mL) to be a various final concentration (2.5 × 10 -7 -2.5 × 10 -3 M).
After centrifugation and removal of supernatant, probenecid solution was added again (1 mL). This suspension was seeded to 96-well U-bottom plates (Grainer Bio-One) at 200 μL each (1.0 × 10 4 cells/well). After incubation at 37 °C under 5% CO2 for 5 hours, the plates were centrifuged (200 × g, 5 min) and 100 μL of the supernatant was transferred to a new 96-well F-bottom plate (Grainer Bio-One). The fluorescence (Excitation: 490 nm, Emission: 530 nm) was measured using the Infinite® 200 PRO M Plex (TECAN).
After centrifugation and removal of supernatant, probenecid solution was added again (1 mL). This suspension was seeded to 96-well U-bottom plates (Grainer Bio-One) at 200 μL each (1.0 × 10 4 cells/well). After incubation at 37 °C under 5% CO2 for 5 hours, the plates were centrifuged (200 × g, 5 min) and 100 μL of the supernatant was transferred to a new 96-well F-bottom plate (Grainer Bio-One). The fluorescence (Excitation: 490 nm, Emission: 530 nm) was measured using the Infinite® 200 PRO M Plex (TECAN).