Trypan blue exclusion

The chicken hepatocellular carcinoma cell line (LMH) was used for virulence assays, since it provided a suitable system for the in vitro pathogenicity testing of T. gallinae [14 (link)]. LMH cells were cultured in RPMI-1640 supplemented with 10% FBS, 40,000 IU/ml penicillin and 40 mg/ml streptomycin (Gibco^™, Thermo Fisher Scientific, California, USA). Cells were incubated in 5% CO₂ and 85–90% humidity at 37°C.
Ten ml of medium with an initial inoculum of 1.4 x 10⁶ cells were cultured in 25 cm² filtered cap flasks (Nunc^™, Thermo Fisher Scientific, California, USA). The cultures were split every 3–4 days by adding 2 ml of a trypsin solution (0.05% of trypsin in 0.53 mM EDTA, Thermo Fisher Scientific, California, USA) for 3–5 min followed by washing of the cells in 50 ml of sterile PBS (pH 7.2). After centrifugation at 1,300 rpm for 5 minutes the cells were suspended in 10 ml of fresh medium. To assess cell viability, trypan blue exclusion was used (Sigma-Aldrich, St. Louis, Missouri, USA).

At the end of the expansion phase in the 3D culture system, cells were extracted by substituting the CM with a solution of 0.3% collagenase (collagenase) and perfusing the ceramic constructs for 40 min followed by 0.05% trypsin/0.53 mM EDTA solution (trypsin) for additional 15 min both at 400 µm per second. Extracted cells were subsequently sorted using anti-CD45-coated magnetic beads (Miltenyi Biotec, Auburn, CA), according to the manufacturer's instructions. 2D-expanded cells were retrieved by using the same enzymatic solutions, i.e. collagenase for 40 min and trypsin for 5 min. The fraction of dead cells, preliminarily assessed by assessed by Trypan blue exclusion (Sigma, Switzerland), was negligible (less than 3%), with no obvious differences between the experimental groups. Both CD45⁺ and CD45⁻ viable cell populations were assessed for the ability to form fibroblastic colonies. The CD45⁻ populations were further characterized by flow cytometry, gene expression by means of microarray analysis and quantitative real-time (QRT) PCR or tested for the multilineage differentiation capacity, as described below.

Heparinized total blood was collected through venipuncture and centrifuged for 8 h with Ficoll-Paque ™ specific gravity gradient 1077 (GE Healthcare). The PBMCs were collected, rinsed three times with phosphate buffer saline, and counted via trypan blue exclusion (Sigma-Aldrich, St. Louis, USA) in a Neubauer chamber. The cells were resuspended in aliquots of 2 x 10⁶ PBMCs/mL in RPMI 1640 medium (GIBCO, Life Technologies, UK) containing 10% of inactivated fetal bovine serum (GIBCO, USA),followed by the addition of phytohemagglutinin in a concentration of 5 μg/mL (PHA-P, Sigma-Aldrich, St. Louis, MO, USA) [37 (link)].
Black, 96-well, polystyrene plates (Greiner Bio-One, USA) were used to incubate 100 μL of PBMC suspension and C. zeylanicum Blume EO (31.25–2,000 μg/mL) at a ratio of 1:1 in culture medium at 37°C, in a humidified atmosphere, and with 5% CO₂ for 24 h. Next, the cell viability was measured using the alamarBlue® fluorescence protocol (Bio-Rad, Hercules, CA, USA). The percent viability was calculated as follows: $\begin{array}{c}\%\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{y}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}\\ =\left[\left(\frac{\mathrm{F}\mathrm{I}\hspace{0.17em}\hspace{0.17em}\mathrm{590}\hspace{0.17em}\hspace{0.17em}\mathrm{o}\mathrm{f}\hspace{0.17em}\hspace{0.17em}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\hspace{0.17em}\hspace{0.17em}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{F}\mathrm{I}\hspace{0.17em}\hspace{0.17em}\mathrm{590}\hspace{0.17em}\hspace{0.17em}\mathrm{o}\mathrm{f}\hspace{0.17em}\hspace{0.17em}\mathrm{n}\mathrm{o}\mathrm{n}\text{-}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\right)\left(\mathrm{100}\right)\right],\end{array}$ where FI 590 = the fluorescence intensity at 590-nm emission (560-nm excitation).

At indicated times, mice were sacrified and blood, spleen, lymph nodes (inguinal, brachial and axillary) and thymus were harvested. Single cell suspensions were generated from spleen, lymph nodes and thymus by homogenization using a 100 μm cell strainer on a petri dish. Erythrocytes were lysed with lysis buffer for 5 min and then the cells were washed with PBS containing 3% FBS. Cells were washed again with PBS containing 3% FBS or with complete medium before use in the assays described below. Viable cells were counted by trypan blue exclusion (Sigma-Aldrich, St.Louis, MO).