THE PROTOCOL:
(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 week 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 (see Recipes).
(3) Warm apical and basal solutions to 37 degree celsius in a small waterbath on your bench.
(4) Pour 300 mL basal solution in a 400 mL beaker, set the beaker on a
37 degree celsius heat block on your bench.
(5) 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.
(6) Take cells from incubator and put the plate on a 37 degree
celsius heat block on your bench. Label Transwells (#1, #2, #3...).
(7) Take Transwell #1 from the cell culture plate, hold the cup over the beaker with the basal side slightly submerged
into the basal solution. Be sure to avoid dipping too deep or tilting the Transwell cup and NEVER let the basal solution get
into the apical well.
(8) With one hand holding
the Transwell cup, use the other hand to pipet 10 mL apical solution from the bottle that has been sitting in the 37 degree
waterbath on your bench.
(9) 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. Be sure to keep
the bottom of the Transwell cup in the basal solution at all time but NEVER let the basal solution to flow into the apical
well.
(10) Rinse the Transwell twice more with
10 mL apical solution.
(11) 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.
(12) Quickly remove 0.5 mL apical solution from the top of the Transwell cup. That should leave 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.
(13) 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.
(14) Mark down the START TIME for Transwell #1.
(15) Repeat procedure for Transwell #2, #3....
(16) After all Transwells are set up, place the 12-well plate
in a 37 degree celsius humidified incubator.
(17)
Allow flux for 2 hours [this is the length of flux time].
(18) 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.
(19) Remove Transwell #1 from the well.
(20) 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. Store the apical sample #1
in an eppendorf tube.
(21) Collect the rest of
apical samples #2, #3...
(22) Store the sample
overnight at 4 degree celsius or proceed to ION QUANTITATION ASSAYS.
