199 culture medium

A primary cell culture derived from pleomorphic adenoma was established. Briefly, neoplasm fragments were chemically digested with trypsin and then mechanically dissociated with a Pasteur pipette. Cells were placed in 25 cm² culture flasks with culture medium 199 (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), and incubated in a humidified atmosphere of 5% CO₂ at 37°C. AP-1 cell line was derived from a subculture of the primary cell culture.

The cells were incubated at 37°C in culture medium 199 (Gibco) for 2, 24, and 72 hours, subsequently washed with PBS to remove the medium, and stained with the acridine orange vital dye. The percentages of viable cells and cells in the death process were determined using fluorescence microscopy.
To evaluate the cellular proliferation index in coculture (PMN and MCF-7 cells), it was incubated with propidium iodide (PI), and subsequent fluorescence detection allowed for assessment of the number of nonvital cells (first measurement). Thus, the cells were treated with melatonin incorporated or not to microemulsion and put in 24-well culture plates. The cells were maintained in culture for 72 hours, stained with PI, and had access to total DNA, leading to total cell population counts (second measurement). The fluorescence of the cells was analyzed by flow cytometry (FACSCalibur system; BD, San Jose, USA). The difference between these two measurements calculated the number of viable cells. The cellular proliferation index was calculated using the number of viable cells treated/number of viable cells not treated × 100 [26 (link)].

Leishmania (Leishmania) amazonensis (MHOM/BR/1989/166MJO) was used in promastigote forms, in the stationary growth phase (day 5 of culture). The parasites were obtained from popliteal lymph nodes of L. amazonensis-infected BALB/c mice and maintained in 199 culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 10 mM HEPES Biological Buffer (AMRESCO), 0.1% human urine, 0.1% L-glutamine (SYNTH), penicillin (10 U/mL) and streptomycin (10 μg/mL) (Gibco), and 10% sodium bicarbonate (SYNTH). Cell cultures were incubated at 25°C in 25 cm² flasks. All parasites were from a culture that was serially passed for less than 5 weeks.

The samples were stored in a sterile plastic tube and centrifuged for 10 minutes at 160 G under refrigeration at 4°C, separating the colostrum into three distinct phases, cellular “button,” intermediate aqueous phase, and lipid supernatant, according to Honorio-França [21 (link)]. Next, the cell button was resuspended in 199 culture medium (Gibco) and separated in a density gradient with Ficoll-Paque (Pharmacia) for 40 minutes at 160 × g at a temperature of 4°C. Then, the cells were adjusted to a final concentration of 2 × 10⁶ cells/ml by light microscopy.

1.5-days-old embryos were electroporated with a pNLS-EGFP-L2-PCNA (Leonhardt et al., 2000 (link)) vector, to distinguish the G2/M/G1 phases of the cell cycle, at 0.5 µg/µl. 6 hr later, embryos were dissected, fluorescent neural tubes were transferred to a tissue chopper (Mc Ilwain) and 100 µm thick transverse sections were sliced. Sections were collected in 199 culture medium (GIBCO) and were sorted out under a fluorescence microscope to control tissue integrity and the presence of isolated fluorescent cells along the dorso-ventral axis. Each slice was imbedded into 10 µl of rat type I collagen (Roche; diluted at 80% with 1X MEM (GIBCO), 1X GlutaMax (GIBCO) and neutralizing bicarbonate (GIBCO)). Four neural tube-containing collagen drops (5 µl) were distributed on a 35 mm glass-bottom culture dish (IBIDI). Collagen polymerization was performed at 38°C for 30 min and 1.5 ml of complete culture medium (199 medium, 5% FCS, 1X GlutaMax, Gentamicin 40 µg/ml) was gently added. For time-lapse, images were acquired on an inverted microscope (Leica inverted DMI8) equipped with a heating enclosure (set up at 39°C), a spinning disk confocal head (CSU-X1-M1N, Yokogawa) a SCMOS camera and a 63X oil immersion objective (NA 1,4–0,7). We recorded 40 µm thick z stacks (2 µm z-steps) at 5 min intervals. IMARIS and ImageJ software were used for image processing and data analysis.

Leishmania (Leishmania) amazonensis (MHOM/BR/1989/166MJO) was used in promastigote forms, in the stationary growth phase (day 5 of culture). The parasites were obtained from popliteal lymph nodes of L. amazonensis-infected BALB/c mice and maintained in 199 culture medium (GIBCO) supplemented with 10% fetal bovine serum (FBS) (GIBCO), 1M HEPES Biological Buffer (AMRESCO), 1% human urine, 1% L-glutamine (SYNTH), penicillin (10 U.mL^-1) and streptomycin (10 μg.mL^-1) (GIBCO) and 10% sodium bicarbonate (SYNTH). Cell cultures were incubated at 25°C in 25-cm² flasks. All parasites were from a culture that was serially passed for less than 5 weeks.