(1) Plate cells at ~30% to 50% confluency on 1 cm square Transwell-clear (Costar). Plate enough Transwells to perform
flux experiments in triplicates. After the cells reach confluency, allow another weelk for them to fully differentiate and
polarize. Important: feed cells generously after they reach confluency (see Epithelial Cells).
(2) Prepare fresh apical and basal solutions on the day of flux experiments. Apical solution is
regular growth media without serum. Basal solution is media without serum plus the radioactive tracer. Add radioactive
tracer to a final concentration of 10 microMolar to the basla solution [this is the DRIVING FORCE].
(3) Warm apical and basal solutions to 37 degree celsius in a small waterbath on your bench.
(4) Pipet 2 mL basal solution to each well of a 12-well plate, cover the
plate with lid and put the plate on a 37 degree celsius heat block on your bench. LABEL "RADIOACTIVE".
(5) Take cells from incubator and put the plate on a 37 degree celsius heat block on your bench.
Label Transwells with cells (#1, #2, #3...)
one hand holding the Transwell cup, use the other hand to pipet 10 mL apical solution that has been sitting in the 37 degree
waterbath on your bench.
(7) Gently and slowly pipet the entire 10 mL apical solution
onto the top edge of the inside wall of the Transwell cup. Allow the apical solution to flow into the apical cup and dilute
the media already in the cup. Let the overflow run into the beaker.
(8) Rinse the Transwell twice more with 10 mL apical solution.
(9) The Transwell cup should have fluid up to its rim. Quickly remove Transwell from the beaker. Gently remove excess
fluid from the outside of the Transwell cup with a kimwipe. Be sure not to touch the bottom of the filter.
(10) Quickly remove 0.5 mL apical solution from the top of the Transwell
cup, leaving ~ 0.5 mL apical solution in the Transwell cup for the fllux experiment. Be sure not to tilt the cup to expose
cells to air.
(11) Immediately put the Transwell cup into a well of the
12-well plate on the 37 degree celsius heat block on your bench that has already warm basal solution in the wells.
(12) Mark down the START TIME for Transwell #1
(13) Repeat procedure for Transwell #2, #3....
(14) After all Transwells are set up, place the 12-well plate in a 37 degree celsius humidified incubator.
(15) Allow flux for 2 hours [this is the length of flux time].
(16) Prepare scintillation vials for counting radioactivity. Add 5 mL
scintillation cocktails to each glass vials.
At the end of two hours, take the 12-well plate out and put it on a 37 degree celsius heat block on your bench.
(18) Remove Transwell #1 from the well.
(19) Tilt the Transwell cup slightly to collect the entire apical solution using a 1 mL pipetman.
It is alright to expose cells to air since it is the end of experiment. Pipet apical sample #1 to a scintillation vial which
has already 5 mL cocktail in it (STEP 16).
Collect the rest of apical samples #2, #3...
Count radioactivity. Remember to add a blank sample with apical media (~ 0.5 mL) and a Standard sample for calculating tracer
concentration. Add 10 microL of basal solution to 0.5 mL apical solution for the Standard sample.
(23) To calculate flux value, see Cpm-Flux Conversion.