Sodium Fluorescein Permability Assay*
The following is a specific protocol adapted from: Tchao, Ruy, Progress in In-Vitro Toxicology 6 (2). It is a MDCK cell monolayer integrity test performed on Millicell-HA cell culture insert using sodium fluorescein. This assay is suited to score the irritancy of compounds and mixtures that are miscible in water, most commonly soaps and detergents. These results have been correlated to Draize eye irritancy scores or Average Maximum Draize (AMD) scores.** The assay may be used to establish rank orders of the irritancy of soaps and detergents and may potentially be used to assess and predict irritancy-related compounds.Other diluents may be substituted for less miscible materials. Suggestions include coconut oil, mineral oil, and dimethyl sulfoxide (DMSO). Corn oil, cotton seed oil, soy bean oil, and mineral oil (heavy and light) have shown no adverse effects in this assay. Sodium dodecyl sulfate (SDS) in homogenized mineral oil produced an effect similar to SDS in Hank’s Balanced Saline Solution (HBSS).
This test quantitatively determines the integrity of in vitro MDCK cell to cell junctions in an epithelial monolayer. It has been shown that increased permeability of compounds is related to increased irritancy. The irritancy of unknown samples is determined by comparison to an SDS positive control. This assay also allows evaluation of cell recovery after injury. For example, disruption of tight junctions (without cell detachment and loss) will require two to four hours for complete recovery. If cell detachment and loss occur, the recovery may require several days depending on the severity of the injury. A limitation of the assay is that it can not be used to test compounds of extreme pH (lower than pH 3 or higher than pH 11) due to the buffering capacity of the diluent used. In general, compounds at these extreme pH levels are by definition considered irritants and therefore would not be tested.
*This section contains a protocol for sodium fluorescein. Information on immunofluorescence and Lucifer Yellow is available in the Fixing and Staining section of the Handbook.
**All Draize test scores were obtained from the Soap and Detergent Association. Millipore was not involved in Draize eye irritancy testing.
Materials and Reagents
- 24-well tissue culture plates (3 plates for each compound)
- 12 mm Millicell-HA cell culture inserts.Millipore cat. no. PIHA 012 50
- MDCK epithelial cells
- T-75 tissue culture flasks
- MEM media with 10% fetal bovine serum and gentamycin, 1 µg/mL
- Hanks' Balanced Salt Solution (HBSS)
- Trypsin-Versene™
- Sodium Fluorescein, 1mg/vial
[Adapted from Tchao, Ruy, Progress in In-Vitro Toxicology 6 (2)]:
- Grow MDCK cell line in T-75 culture flask with MEM (10% FBS, 1 µg/mL gentamycin) and pass at weekly intervals using trypsin-versene.
- Place inserts into a 24-well culture plate containing 0.5 mL media per well. One 24-well plate can be used to test a compound at six concentrations in quadruplicate.
- Seed 1.5×105 cells per insert; confluency should occur in 3 days. Test for monolayer before proceeding. During this incubation period the media should be changed daily.
- When cultures are confluent, remove media and rinse inserts with HBSS.
- Place inserts into a new 24-well plate containing 0.5 mL HBSS and a specific amount of the test compound. Incubate at 24°C for 15 minutes.
- Decant solution and wash inserts three times, each with 1 mL HBSS.
- Place inserts into a new 24-well plate containing 0.5 mL HBSS. Add 0.5 mL HBSS containing 0.02% sodium fluorescein to the apical side of the inserts. Incubate at 24°C for 30 minutes.
- Remove inserts and fix for examination via light and electron microscopy.
- Dilute sodium fluorescein solution in each well to 3 mL and measure in a spectrophotometer at 490 nm.
