Tight Junction PROTOCOLS
Tracer Flux Assay
Electron Microscopy
Silver Staining Gels
Ion Flux Assay
Tracer Flux Assay
Cell Culture

The goal of this protocol is to obtain transepithelial flux of radioactive tracers, 14C-mannitol and 14C-ethanolamine.  Larger tracer (3H-Inulin) can also be measured with minor modification (see below).

Permeability Calculation

Cpm-Flux Conversion

Using Radioactive Tracers:

- Order radioactive tracer(s) and keep them in freezer until use to reduce chemical break down. 
- Chemical degradation can generate smaller radioactive compounds that will give erroneous flux results. 
- In general, 14C-Tracers are preferable because covalent bonds formed by 3H are less stable. 
- Shelf live of a 14C-Tracers can be up to 6-9 months if stored properly ( i.e. have not gone through more than 3 freeze-thaw cycles), while 3H-Tracers is probably reliable up to 3-5 months for FLUX experiments. 

Using 3H-Tracers:

- For 3H-inulin, take advantage of its large size and purify it away from its degradation products using a G25 spin column each time before use. This is an essential step because tight junction is usually IMPERMEABLE to 3H-inulin.
- Most published works did not use FRESHLY PURIFIED 3H-inulin, which makes the results questionable (at least less convincing) !! If you don't believe this, try counting activities of the G25 spin column used after you purify the 3H-inulin, there should be enough radioactivity to RUIN YOUR FLUX EXPERIMENTS.


(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...)

(6) With 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.

(17) 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).

(21) Collect the rest of apical samples #2, #3...

(22) 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.

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