Bovine hemin

An 18 nL injection of each product (or PBS as mock control) was performed in the thorax of young females using a Nanoject II (Drummond). Spiroplasma was quantified by qPCR 7 days after the treatment. FAC (Sigma F5879) was injected at 50 mM, BPS (Sigma 146617) at 10 mM and bovine hemin (Sigma H9039) at 10 mM, bovine holo-Tsf (Sigma T1283, Saint-Louis, MO, USA) and human apo-Tsf (Sigma T1147) at 300 µg per fly. Spiroplasma quantification was performed 7 days after the treatment in a least four biological replicates.

The CfC1 genome reference strain of Crithidia fasciculata was used for our experiments. The line was obtained from the laboratory of Dr. Stephen Beverley (Washington University). For these experiments, cells were maintained in complete brain heart infusion medium (BHI) made by dissolving 37 g of powder (Sigma) per liter of water and supplementing with bovine hemin (Sigma) to a final concentration of 20 μg/ml. Cells were passaged every 2–3 days and cultured between 27–28 °C at a density of 10⁵−10⁸ cells/ml in non-treated tissue culture flasks. For cultures of swimming cells, flasks were placed on a rocker, while flasks for adherent cells were left stationary on the incubator shelf. All washes of adherent cultures were performed by pipetting the wash solution directly onto the flask surface, then rocking the flask. Swimming cells were counted by fixing in 0.3% formalin followed by staining with Gentian violet (Harleco) in 0.2 M NaCl, 0.7 mM EDTA. Fixed, stained cells were then counted on a hemocytometer.

C. fasciculata strain CfC1 was grown in brain heart infusion (BHI) medium supplemented with 20 μg/ml bovine hemin (Sigma) at 27°C. Cells grown on a rocker were passaged as nectomonads every 2–3 days to maintain a density between 10⁵ and 10⁸ cells/ml. Cell densities were determined by mixing a small sample of cells with an equal volume of 3% formalin, followed by staining with crystal violet before loading samples on a hemacytometer for counting. To generate adherent haptomonads for imaging, 2 ml of culture at a density of 1 x 10⁷ cells/ml was seeded in a 35 mm poly-L-lysine-coated MatTek dish. The cells were allowed to adhere for 2 hours without rocking. Following 3 washes with BHI, fresh media was added, and dishes were incubated without rocking for 24 hours prior to imaging. To obtain clonal C. fasciculata cell lines, serial dilutions of culture were plated on BHI plates made with 0.65% agarose and supplemented with hygromycin (Amresco) [35 (link)]. After 4–5 days of growth, parasite colonies were transferred with a pipet tip to a standard liquid culture containing hygromycin.