Schneider s media

P. falciparum (strain NF54) was maintained according to standard methods at 37 °C (5% O₂ and 5% CO₂, nitrogen balanced) in 5% hematocrit (O + blood), 10% AB human serum (The Interstate Blood Bank INC, Memphis, TN, USA), RPMI 1640 medium (Cat No. CUS-0645, KD medical, Columbia, MD, USA), and 2.5% sodium bicarbonate⁷⁴ . Two gametocyte cultures were started three days apart using standard methods and an aliquot of each was mixed together when stage V gametocytes were prevalent (day 14–17 of gametocyte cultures) and used to infect day 4–6 old mosquitoes. Infections of laboratory-reared Anopheles (An.) stephensi (Nijmegen strain) were accomplished using a temperature-controlled Hemotek membrane-feeding device (Discovery Workshops, Accrington, UK) and kept at 80% humidity in 26 °C environmental chambers with 10% sucrose supplemented water^{75 (link)}. Additionally, P. falciparum (strains NF54-WT and NF54-GFP) infected An. stephensi mosquitoes were shipped live from John Hopkins University Malaria Research Institute (JHUMRI) insectary and parasitology core (Baltimore, MD, USA) on day 10 post-infection and maintained at 80% humidity in 26 °C environmental chambers with 10% sucrose supplemented water. Salivary glands of mosquitoes 14–16 days after infection were aseptically dissected and collected into Schneider’s media (Cat No. S9895, Sigma-Aldrich, St. Louis, MO, USA)^{76 (link)}.

Flies were euthanized by carbon dioxide narcotization using the Flow Buddy CO₂ System (Flystuff). The MTs were carefully dissected under a stereomicroscope in a Sylgard (Dow Corning)-lined Petri dish using Schneider's media (Sigma-Aldrich). The tubules were mounted on a fresh Petri dish and minutien pins (Fine Science Tools) were used to anchor the ends. Each tubule was incubated in either 200 ml 0.1 mM alendronate-FITC, 0.1 mM notdronate-FITC or PBS for 30 min. Consequently, tubules were washed several times with PBS and then mounted for wide-field fluorescence imaging (Nikon), birefringence microscopy (Nikon), or resonance confocal microscopy (Nikon).
For the MTs expressing RFP, they were first stained with 4′,6-diamidino-2-phenylindole (DAPI) followed by our fluorescent probes alendronate-FITC and notdronate-FITC. The tubules were first imaged with normal light followed by polarized birefringent microscopy and finally imaged with a scanning laser with a confocal microscope. We used a Nikon TE2000 Inverted Microscope and a Nikon Confocal Microscope for imaging.

E.coli strains DH5α (Invitrogen), Bh Houston-1,^{47 (link)}Bh Houston-1 ΔbadA^{24 (link)} and Bh Houston-1 ΔbadA/pNS2P_TrcbadA (this study) were all used for this study (Table 1). E.coli DH5α was grown at 37 °C on either LB agar or liquid LB broth. The Houston-1 strain of Bh used for this study was isolated from an HIV patient.^{47 (link)}Bh was grown on heart infusion agar supplemented with 1% bovine hemoglobin or liquid Schneiders media (Sigma Aldrich, S9895) supplemented with 10% fetal bovine serum for 3 days as described by Riess et al.^{48 (link)} Growth conditions were kept at 5% CO₂ at 37 °C. Bh Houston-1 ΔbadA, a non-polar in-frame deletion mutant of badA has been described by Lima et al., 2014.^{24 (link)}Bh Houston-1 ΔbadA/pNS2P_TrcbadA construction is described as below. Bh Houston-1 ΔbadA/pNS2P_TrcbadA was grown on agar supplemented with kanamycin (50 µg/ml).

Wolbachia was isolated from the ovaries of wAlbB-infected females according to [24 (link)] with the following modifications. Ovaries from 200 wAlbB-infected females were dissected on ice and suspended in 50 μL of ice-cold Schneiders media (Sigma-Aldrich) in a 1.5 mL eppendorf tube. The ovaries were crushed briefly using a small plastic pestle after which one 3 mm glass bead was added and the suspension vortexed for 2 min. One mL of ice-cold Schneiders media was added to the homogenised and the solution were centrifuged at 4°C for 5 min at 2000 x g. The supernatant was subsequently sequentially filtered through 5 μM and 1.2 μM syringe filters. The resulting filtrate was centrifuged for 4°C for 10 min at 12000 x g. The supernatant was discarded and the pellet resuspended in 50 μL of ice-cold Schneiders media until use. The extraction was repeated with ovaries from W.T. females for use in control injections. Total bacterial counts were estimated using the LIVE/DEAD staining kit (Thermofisher) and counting the live stained bacteria in a hemocytometer.

The L. major Δlcb2 mutant^{24 (link)}, maintained in Schneider’s media (Sigma-Aldrich) with 15% sera (ThermoFisher), were washed and 200 µl, at 1 × 10⁷ ml⁻¹, incubated in serum free media for 60 minutes at 26 °C before the addition of 10 µM of the compounds, in at least triplicate, and further incubation for 18 hours. Subsequently, the reaction was initiated by the addition of BODIPY FL C₅-ceramide complexed to BSA (1.25 µl of 0.5 mM, ThermoFisher). Following further incubation at 26 °C for 1 hour, the lipids were extracted by phase separation in chloroform:methanol:water (10:10:3, 200 µl) and fractionated on HTPLC plates, as previously^{18 (link)}. The fluorescence was read at Ex475/Em520 using a Fuji FLA-3000 plate reader and AIDA Image Analyser software (version 3.52).

Brains of L3 larvae (~120 h after egg laying) were dissected in Schneider’s media (Sigma Aldrich) containing 10% foetal calf serum (ThermoFisher Scientific) and transferred to 50 μl wells of Ibidi Angiogenesis μ-Slides for live imaging. Mutant and control brains were imaged in parallel at 25°C. Z-series with a height of 20 μm and 1-μm spacing were acquired every 30 s using a spinning disk system consisting of a Leica DMi8 microscope equipped with a 63x/1.4 NA oil-immersion objective, a CSU-X1 spinning disk unit (Yokogawa) and an Evolve Electron-Multiplying CCD (EMCCD) camera (Photometrics). The microscope was controlled by Inscoper Imaging Suite software (Inscoper). Images were processed with Fiji (Schindelin et al, 2012 (link)).