Pharmingen

Peritoneal macrophages were infected by L. amazonensis promastigotes always at a ratio of 2 parasites per cell. Axenic cultures of promastigote forms in stationary phase were left in contact with the cells for 6 hours. After, the culture was washed with PBS pH 7.0 to remove promastigotes from the supernatant.
Experiments with pro-inflammatory stimulation used 5 μg/mL of LPS (Sigma-Aldrich, St. Louis, MO) and 2 ng/mL of recombinant mouse IFN-γ (BD Pharmingen^™, BD Biosciences, San José, CA). In experiments with anti-inflammatory stimulation, 2 ng/mL of recombinant mouse IL-4 (BD Pharmingen^™, BD Biosciences, San José, CA) were used. Cells were stimulated 6 hours after infection. The number of cells used in each experiment is described in S1 Table.

Histology analysis was performed for the plasma conditions at 1, 2, 3, 4, 5 L/min and delivery time 4 min. After the treatment, patch soaked with FITC labelled dextran was applied on the tissue as described before. To determine the depth and extent of skin damage the region of the skin exposed to the treatment was excised 3 h post-treatment. The excised skin was fixed in Zn fixative (BD Pharmingen^TM, BD Bioscience, San Diego, CA) for 24 h and then stored in 70% ethanol until embedding in paraffin. 5 μm thick sections were cut and stained with haematoxylin and eosin or Masson’s trichrome. The slides were observed with BX-51 microscope (Olympus, Hamburg, GE) equipped with a digital camera DP72 (Olympus).

Since exposure to UV irradiation and sunlight causes not only skin pigmentation, but also skin cancers, i.e., melanoma- and non-melanoma skin cancer, this study was conducted to determine the cytotoxicity of AG solution, Blank NE and a representative AG-NE against the A375 cells and A-471 cells via apoptosis induction by a flow cytometry technique. Both cells were seeded in 12-well plates at a density of 2 × 10⁵ cells/well/1000 μL and incubated at 37 °C, under 5% CO₂ atmosphere until confluence. They were incubated with the test samples for 24 h. After incubation, the cells were suspended in 1X binding buffer at a concentration of 105 cells/100 µL. Annexin V-FITC (4 µL) and propidium iodide (PI) (4 µL) (BD PharmingenTM, BD Biosciences, San Diego, CA, USA) were added to the cells and incubated for 15 min. Then, the binding buffer (400 µL) was added to each tube containing the cells prior to analysis by a flow cytometry instrument (BD FACSVerse™, BD Biosciences, USA) at an excitation/emission wavelength of 490/520 and 535/617 nm for FITC and PI, respectively. The fluorescent-activated cell sorting (FACS) were plotted and analyzed by BD FACSVerse™ software V. 1.0 (BD Biosciences, USA).

For CD152 labelling (BD Pharmingen^TM, BD Biosciences, San Jose, CA, USA), 1 × 10⁶ cells were washed with 1 mL PBS and incubated with Fix & Perm Medium A solution (Invitrogen^TM, Carlsbad, CA, USA) for 15 min at room temperature in the dark for permeabilization of the cell membrane. The cells were then rewashed with PBS and incubated with Fix & Perm Medium B and CD152 (PE-Cy5) antibody for 30 min at room temperature in the dark. Afterwards, the cells were washed with PBS and fixed with 1% paraformaldehyde (PFA). Cell acquisition was performed using a FACS Calibur cytometer (BD Biosciences, San Jose, CA, USA), and the analysis was performed using FlowJo software (FlowJo, Ashland, TN, USA).

The expression of PCNA and CD31 were estimated by immunochemistry with PCNA and CD31 antibodies. Briefly, the frozen tumor tissues were fixed in acetone and washed twice with PBS. Following, the handled samples were stained with rabbit anti-mouse CD31 monoclonal antibody (1:50; Abcam, Cambridge, UK) and rabbit anti-rat PCNA polyclonal antibody (1:50; BD Pharmingen^TM; BD Biosciences, San Jose, CA, USA). After washing twice with PBS, samples were stained with secondary antibody combining with FITC or Rhodamine. Finally, we surveyed the cells possessing-positive property under microscope (×400) and counted automatically the number of capillaries in 5 randomly selected fields.