Ti e widefield microscope

Samples for all imaging experiments were imaged on a Nikon Ti-E spinning disc confocal microscope equipped with Nikon Elements software, Ti-E perfect focus system, Yokogawa CSU-X1 spinning disc head, Andor 888 Ultra EMCCD camera and Oko Labs enclosed environmental chamber set at 37 °C.
Samples for each type of imaging experiment except immunofluorescence experiments were also imaged on a Nikon Ti-E widefield microscope equipped with Nikon Elements software, Ti-E perfect focus system, Andor iXon3 EMCCD camera, Sutter Instruments LD-LS/30 xenon arc lamp, and Sutter Instruments Lambda 10-3 filter changer.
For electrical stimulation, an IonOptix Myopacer cell stimulator was equipped with a custom set of platinum wire electrodes. Stimulations were performed with 30 V bipolar waveform 10 ms pulses at 5 Hz for a duration of 1 minute, unless specified otherwise.
RNA extraction for next-generation sequencing was performed using a Promega Maxwell RSC Instrument.
Quantitative PCR was performed using a BIO-RAD CFX384 Real Time PCR Detection System instrument.

BHK cells co-expressing GFP-tagged Ras anchors and the empty vector pC1 or RFP-tagged Ras anchors were grown to ∼85% confluency and washed with 2× Hepes buffer. The cells were then incubated in Hepes buffer containing 2 mM (N-ethyl maleimide [NEM]) for ∼90 min to induce blebbing (83 (link)). GPMVs containing GFP- and RFP-tagged Ras anchors (or GFP-tagged Ras anchors with pC1) were then incubated in Hepes buffers containing various percentages of deionized water (hypotonic conditions) or concentrations of NaCl (hypertonic conditions) for 5 min.
GFP fluorescence was visualized using a Nikon TiE wide-field microscope using a 60× oil-emersion PLAN-Apo/1.4 numerical aperture lens (25 (link),26 (link)). The fluorescence lifetime of GFP was measured using a Lambert FLIM unit attached to the wide-field microscope. GFP was excited using a sinusoidally stimulated and modulating 3-W 497-nm light-emitting diode at 40 Hz. At least 20 vesicles were imaged and the fluorescence lifetime values were pooled and averaged. Statistical significance was evaluated using one-way ANOVA, with * indicating P < 0.05.

All mouse procedures were performed in accordance with the UK Animals (Scientific Procedures) Act (PPL 70/7185) and approved by the Imperial College Animal Welfare and Ethical Review Body (AWERB). The ability of test compounds to inhibit P. berghei early mosquito stage development was tested in the Ookinete Development Assay^{37 (link)} with a few exceptions. Anaesthetised 6- to 8-week-old female Tucks Ordinary (TO) mice infected with P. berghei strain CTRPpGFP^{38 (link)} were rapidly exsanguinated by cardiac puncture and 500 µl of blood diluted in 25 ml of ookinete medium supplemented with 20% foetal bovine serum. Immediately the blood/medium mixture was dispensed by multichannel pipette into 384 well plates containing pre-plated test compounds. 10 µM cycloheximide and DMSO were used as the positive and negative controls, respectively. Plates were stored in the dark in a humidified incubator at 18 °C for 24 h. Ookinete production was imaged by fluorescence microscopy of GFP expression under the ookinete-specific CTRP promoter using a Nikon Ti-E widefield microscope and ×10 objective. Ookinete production was quantified using ICY and percentage inhibition calculated as above with reference to positive and negative controls.

Primary cortical neurons were isolated from E15 mouse embryos dissociated in 0.05% trypsin-EDTA (Thermo Scientific) at 37 °C for 12 min. 320,000 cells/ml were plated on poly-D-lysine (0.125 mg/ml, Sigma Aldrich) and lamin (5 µg/ml, Corning) coated 96-well glass plates and grown at 37 °C and 5% CO₂ in Neurobasal medium (Thermo Fisher) supplemented with 2% B27 and 1% Glutamax (Thermo Scientific). Four DIV neurons were transfected with 0.2 µl Lipofectamine 2000 (Thermo Scientific) and 100 ng DNA following manufacturer’s recommendations. A ratio of 4:1 was used for the GFP and S-mCherry vectors. Thirsty-six hours after transfection, Leptomycin B (10 mg/L) was added to the culture medium immediately before image acquisition. Cells were imaged using a Nikon TiE widefield microscope equipped with temperature- and CO₂-controlled environmental chamber. Movies were acquired with a ×20 lens at a rate of 1 frame every 1 or 2 min for 1 h. For rescue experiments, neurons were fixed immediately after acquisition and stained with V5 antibody to detect V5-PFN1 expression.

Motor neurons were co-transfected as outlined above with green fluorescent protein (GFP) and V5-tagged PFN1 constructs in a 1:2 ratio. KPT-276 (50 nM) or equal volumes of DMSO were added to the culture medium 1 day after transfection and maintained throughout the experiment for a total of 3 days. Cells were fixed and stained to detect V5-PFN1 expression 4 days after transfection. Cells were imaged as individual focal planes using a ×10 lens. GFP was used to identify transfected cells and highlight the whole cell structure. For live imaging of axon outgrowth, KPT-276 (50 nM) or equal volumes of DMSO were added to the culture medium 1 day after transfection and maintained throughout the experiment for a total of ~18–24 h. Cells were imaged at 3 DIV using a Nikon TiE widefield microscope equipped with temperature- and CO₂-controlled environmental chamber. Movies were acquired with a ×20 lens at a rate of 1 frame every 10 min for 1 h.