Encyclopedia of Experiments: Biology
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Harvest the adherent HEK293T cell culture by trypsinization, and re-suspend the cells in a dedicated complete growth medium. Plate the cells onto a clear bottom, white side, 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls, including replicates. Then, culture the cells overnight in standard conditions.
On the next day, transfect the cells with the desired combinations of expression plasmids. Dilute the plasmids in a serum-free medium to 6.25 nanograms per microliter for each construct, and add the lipid-based transfection reagent at an appropriate lipid-to-DNA ratio.
Incubate the plasmids according to manufacturer's instruction. Then, add 8 microliters of the lipid-DNA mixture to the designated wells. Mix the content of the plate with gentle rotation, and culture the cells for 20 to 24 hours in standard conditions.
On the next day, replace the conditioned medium in each well with 100 microliters of a serum-free medium, making sure that the cells do not detach upon medium exchange.
Just before measuring luminescence, mix one volume of furimazine with 19 volumes of a dilution buffer. Add 25 microliters of the furimazine working solution into each well, and gently mix the plate by hand, or with an orbital shaker.
Insert the plate into a luminescence microplate reader, and equilibrate the plate at 37 degrees Celsius for 10 to 15 minutes. Select the wells to be analyzed, and read luminescence with an integration time of 0.3 seconds. Continue to monitor luminescence for up to two hours when required.
