Measurement of viability after single application: MTT test Step 2 (Day 1)

Note: The MTT is sensitive to light, to be handled under a hood, light off.

1. Take a 24-well plate and place freshly prepared 500 µL of the MTT solution (2 mg / mL in 1X PBS) in each well.
2. Test the parallel wells for absence of interference of the MTT with the test product (s): place a small quantity of product in the bottom of the well (approximately 10 µL or 10 mg) then add 500 μL of the MTT solution (2 mg / mL) without trying to homogenize. The aim is to test whether the test product visually stains in the presence of MTT.
3. Incubate for 20 h ± 2 h.
4. Take each skin disk with forceps and rinse with 1ml of 1X PBS (maintained at room temperature) in order to remove product residues. If it is difficult to remove the product during the test, perform 1 to 2 additional rinses and indicate, if the case arises, the wells where the explants are not properly rinsed.
5. Afterwards sponge on absorbent paper. Note: In order to avoid any cross contamination, the explants are "drained" on different areas of the absorbent paper.
6. Then place each skin explant per well containing the MTT solution (epidermis oriented towards the bottom of the MW well) and incubate the plates as well as the interference control plate for 4 h, at +32 °C ± 1 ° C in the CO2-free oven with a box filled with water to maintain humidity> 60%.
7. After 4 hours of incubation:
   1. Observe the wells of the MTT interference control and note the observations: absence or presence of purple coloration.
   2. For the wells with the skin disks, remove the MTT solution using a pipette
   3. Distribute 2 mL of isopropyl alcohol in each well.
   4. Place an unstretched parafilm film on each plate, replace the cover and place the plates for at least 17 h at + 4 ° C