To prepare a master mix of the working solution, dilute 25 microliters of the WST-1 reagent and 225 microliters of medium for each well. Next, pipette 250 microliters of the master-mix working solution of WST-1 for each well, and incubate the plate at 37 degrees Celsius for one hour. Ensure that the PCLS are fully covered by the WST-1 reagent during incubation.
Afterward, place the plate on an orbital shaker at 200 rpm, and shake carefully for 30 seconds to ensure thorough mixing of the WST-1 reagent. Then, pipette 100 microliters of the supernatant from each well of the 24-well plate to a new flat bottom 96-well plate in duplicates. Measure the absorption of each well at 450 nanometers using a microplate reader, and subtract the absorption at 630 nanometers reference from that at 450 nanometers.
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