Fa 45 30 11 rotor

The pharmacokinetics of 2-PAM in the blood of Wistar rats was studied for unmodified transfersomes after transdermal administration. A gel form of transfersomes (0.6 g) was applied to the shaved areas (2 × 2 cm) on the backs of rats. Blood was taken at 6, 12 and 24 h after transfersome application. Rats were first deeply anesthetized by isoflurane inhalation. Blood samples were collected and mixed with 50 ME of heparin per 1 mL of blood and centrifuged at 8000 rpm for 8 min at 4 °C using Eppendorf 5430R centrifuge with FA-45-30-11 rotor (Eppendorf AG, Hamburg, Germany) to get plasma. There were 6 rats at each time point. The 2-PAM extraction procedure from plasma is described in [51 (link)]. The calibration curves were linear in the range 10.0–300.0 ng/mL (Figure S6).

HEK293T cells were harvested at ~80% confluency by gently pipetting with PBS. Cells were centrifuged at 500 ×g and the pellet was weighed and frozen at −80°C until use. Cells were thawed, resuspended in 2 vol of homogenization buffer (250 mM sorbitol, Tris-HCl, pH 7.4, 137 mM NaCl) containing protease inhibitor cocktail (1mM 4-aminobenzamidine dihydrochloride, 1 µg/ml antipain dihydrochloride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml chymostatin, 1 mM phenymethylsulfonly fluoride, 50 µM N-tosyl-L-phenylalanine chloromethyl ketone and 1 µg/ml pepstatin) and passed 7–15 times through a 22 gauge needle until >80% of cells were disrupted, as assessed by microscopy and trypan blue staining. The homogenized cells were then centrifuged at 1500 ×g and the supernatant fraction was centrifuged at 15,000 ×g using a FA-45-30-11 rotor and Eppendorf 5430 R centrifuge (Eppendorf, Hamburg, Germany). The supernatant fraction was centrifuged again at 55,000 RPM in a TLS-55 rotor and Optima Max XP ultracentrifuge (Beckman Coulter) to generate the cytosol fraction (~5 mg/ml). The 15,000 ×g pellet fraction was resuspended in 2 packed cell vol homogenization buffer and an equal vol of 1 M LiCl. The membranes were then centrifuged again at 15,000 ×g and resuspended in 1 original packed cell vol to generate the membrane fraction.

For long-term maintenance, Lyophyllum sp. strain Karsten and T. asperellum 302 were maintained in autoclaved distilled water. For routine use, the fungi were refreshed once a month on oat flake agar (OFA) plates. For analysis of their respective secretomes, both fungal strains were first grown for 48–72 h in oat flake medium at 28°C with shaking. The well-grown fungal cultures were then centrifuged at 2,655 ×g (FA-45-30-11 rotor, Eppendorf) for 10 min and the pellets washed twice with M9. M9 medium supplemented with 1% [w/v] sodium propionate (pH 4.8) was then used for fungal growth for the preparation of fungal secretomes. Thus, Erlenmeyer flasks containing 50 mL of medium were inoculated with the respective strains and incubated for two weeks at 28°C, with shaking.