Cellometer vision cell counter

The fluorescent substrate calcein AM was used to characterize the transport activity of WT-MRP2 and variants in Flp-In 293 cells (18 (link), 20 (link)). In brief, cell suspensions (prepared in medium, 500,000 cells/ml) were loaded with calcein AM (0.5, 2 or 10 μM) for 30 min at 37°C, 5% CO₂ (uptake period). After washing with culture medium, cells were incubated in culture medium for 60 min to allow calcein AM efflux from the cells (efflux period). Following washing and centrifugation, the cell pellet was suspended in phosphate-buffered saline (PBS) and relative fluorescence units (RFU) for each cell was quantified on a Cellometer Vision cell counter using filter cube VB-535-402 (excitation/emission: 535/402 nm) (Nexcelom Bioscience LLC, Lawrence, MA).

Cells were treated with vehicle, VPA (5mM), apicidin (0.5μM), or
SAHA (10μM) for 24 h and then re-seeded in a 96-well plate to undergo
transporter efflux assays as described in a previous protocol [39 (link)]. Briefly, hCMEC/D3 cells (n=4) were incubated for
30 min in medium containing the MDR1 fluorescent substrate rhodamine 123
(7.5μM) in the presence or absence of the functional inhibitor, verapamil
(100μM) (uptake phase). Cells were then washed,
centrifuged, resuspended, and incubated for 2 h in substrate-free medium in the
presence or absence of the inhibitors (efflux phase). At the
end of the efflux phase, cells were washed and re-suspended in cold PBS and
evaluated for retention of rhodamine 123. Intracellular fluorescence intensity
was quantified in relative fluorescence units (RFU) using the Cellometer Vision
cell counter (Nexcelom Bioscience LLC., Lawrence, MA) with the filter cube
VB-595–502 (Excitation/Emission: 525nm/595nm). The average for each
treatment group was determined by the average fluorescence values of four
independent samples.