Neubauer counting chamber

To determine the proliferation rate of HUVECs on coated ePTFE grafts, cells were seeded to 24-well plates (Corning, USA) in a concentration of 5 ​× ​10³ ​cells/well. Every 24 ​h for three days, cells were detached with trypsin (Gibco, USA) and counted manually with a Neubauer counting chamber (Sigma-Aldrich, Austria).

A pure culture of an indigenous fungal strain G. lucidum IBL-05 available in Industrial Biotechnology Laboratory, Department of Biochemistry, University of Agriculture; Faisalabad was raised on potato dextrose agar (PDA) slants at pH 4.5 and 30 °C. Kirk’s basal medium along with 1 % (w/v) Millipore filtered sterile glucose solution was used as inoculum medium. The medium pH was adjusted to 4.5 (HCl/NaOH), sterilized (Sanyo, Japan) at 121 °C and 15 psi for 15 min. G. lucidum IBL-05 spores were transferred to the sterilized inoculum medium under sterile conditions in laminar flow (Dalton, Japan). The inoculated flask was incubated at 30 °C for 5 days in an orbital shaker (Sanyo-Gallenkamp, UK) with continuous shaking (120 rpm) to obtain homogenous spore suspension (~1×10⁷ spore/mL), counted by hemocytometer (Neubauer counting chamber) (Sigma-aldrich, USA) [5 (link)].

Apoptosis was determined using FACS after staining with Annexin V-FITC (Enzo Lifesciences, Farmingdale, NY) and propidium iodide (Carl Roth, Karlsruhe, Germany) or by counting stained (dead) and unstained (living) cells with a Neubauer counting chamber after incubation with trypan blue (Sigma-Aldrich). A detailed protocol has been described previously (39 (link)).

To measure the enzymatic activity in the SmF, 40 mL of culture medium (see above for medium used) was inoculated with 1 × 10⁶ spores/mL of the corresponding strain in 125 mL Erlenmeyer flasks. The culture was incubated at 28 °C on an orbital shaker at 150 rpm. The samples were collected in triplicate every 24 h for 5 d using a Neubauer Counting Chamber (Sigma-Aldrich, Darmstadt, Germany).
For the SSF, a corncob, 16 mesh size, was used as the substrate. Before use, the corncob was washed three times with hot bi-distilled water, followed by two washes with cold distilled water, and dried at room temperature for 24 h followed by incubation at 50 °C for 48 h. Five grams of the substrate was placed in a 125 mL flask with a 75% humidity. The substrate was inoculated with 1 × 10⁶ spores/g of dry substrate. All flasks were incubated at 28 °C for 5 d in triplicate.

Thymocytes were isolated by grinding thymi through a 50 μM mesh or cell strainer in the presence of 2% FBS PBS. For complement-mediated enrichment of mature thymocytes, the cell suspension was then incubated with anti-CD24 (I) (clone J11d—in house production) on ice for 30 min. Rabbit complement (GTI Diagnostics, Wisconsin, USA) and 70 μg/mL deoxyribonuclease I (DNase; Roche) were then added and allowed to incubate for another 30 min at 37 °C. Thymocytes were washed with 10% FBS/RPMI, then overlayed onto Histopaque-1083 (Sigma), and centrifuged at 1000 × g for 10 min, with no brake, at room temperature. Cells at the interphase were recovered, centrifuged at 350 × g for 4 min at 4 °C and washed with 2% FBS/PBS, or 10% FBS/RPMI for cell culture. All cells were quantified in a Neubauer counting chamber (Merck), with dead cell exclusion by trypan blue solution 0.4% (Sigma).

Co-cultures were set up by first seeding 20-mm diameter glass bottom (0.17-mm thick coverslip) cell culture (treated) dishes (NEST 801001) with 5 × 10⁵ CFUs of actively growing A. castellanii strain Neff trophozoites, previously quantified with the aid of a Neubauer counting chamber (Blaubrand, Merck), which were incubated at 30°C for 1 h in a 1.6-mL salt solution of the defined MM-MOPS medium (133 (link)) plus 0.8% (wt/vol) D-glucose (MMsalts-MOPS-Glc, pH 7.2). Stenotrophomonas maltophilia Sm18 (54 (link)) was grown in LB medium at 30°C. Overnight cultures were washed once in fresh LB, diluted to an OD₆₀₀ ≈0.7, and incubated at 30°C until reaching an OD₆₀₀ ≈1.0, equivalent to ~4.8×10⁸ CFUs/mL. Dilutions of the Sm18 culture were quantified using a Neubauer counting chamber, and 100-µL suspensions at the proper concentration of cells were added to each cell culture dish to reach the desired multiplicity of infection (MOI 12.5, MOI 25, MOI 50, and MOI 100) and centrifuged at 500 × g for 5 min at 30°C to synchronize phagocytosis.

Fermentation protocol was conducted as per the method of Starzyńska-Janiszewska et al. [29 ] with slight modification. Rhizopus oligosporus fungus (ATCC 48010) was incubated on potato dextrose agar (PDA) at 25°C for one week till formation of black spores. The mature spores were then suspended in a mixture containing sterile saline solution at concentration of 1 g/100 mL, 0.01% Tween 80, and peptone at concentration of 0.001 g/100 mL under carefully sterilized conditions. The spore density required for fermentation (10⁵/mL) was prepared using Neubauer counting chamber (Merck, Darmstadt, Germany). Prewashed red quinoa seeds were cooked for 20 minutes in acidified tap water (1 : 3°w/v, pH = 4) and then dried on sterilized filter paper. R. oligosporus spores were inoculated and mixed with one gram of the dried seeds at density of 10⁵/g in sterilized and sealed Petri dish and left incubated at 35°C for germination of spores. Following spore germination, the temperature was then cooled to 30°C, and incubation continued to 48 hours to complete the fermentation process. The fermented quinoa seeds were finally blended in sterile distilled water (1 g/mL), and the resulting suspension was left at −20°C one day before use.