Treatment of explants by single application Step 1 (Day 1)
Each skin disk should have approximately 750 µL media (6cm dish) or 200 µL media (24h well plate) . Add media if there are losses during the transportation.
Before any sampling, homogenize the test products well.
Add 25 µL or 25 mg of test sample on filter paper disks 8mm.
using a P100 pipette for liquid products.
using an M100 pipette for thick or viscous products.
The test product is applied to the filter paper disc while the latter is held using forceps, and thereafter applied directly to the implant, (product against the epidermis) thus ensuring semi-occlusion
If the product is solid: finely cut a coverslip with a diameter of approximately 8 mm and place it directly on the implant, thus ensuring semi-occlusion.
If the product is a powder: weigh a quantity of 25 mg in a 1.5 mL Eppendorf tube and deposit the powder by inversion on a paper disc previously moistened with 25 µL of demineralized water, which is applied directly to the 'explant.
If the product contains volatile ingredients (eg: alcohol, perfume, etc.), evaporate it by shaking the filter paper for 15s, then after application to the explant, leave the Petri dish open under the hood for 60 min. Afterwards add 250 μL of complementary culture medium to compensate for the loss of medium due to evaporation, making sure to respect the time of 60 min for each dish.
Before closing the Petri dishes, make sure that the culture medium permeates the filter paper.
The skin disks are incubated at +32°C ± 1 ° C for 20h ± 2h in the incubator without CO2 with a pan filled with water to maintain humidity > 60%.
Check with a hygrometer and record the measurement.
For maximum humidity, position the Petri dishes in the center of the oven tray.
