Axiocam icc1 digital camera

Blood containing approximately 50,000 parasites was injected intraperitoneally (i.p) into mice to initiate infections. Four to five days post infection, exflagellation and ookinete conversion were examined as described previously^{42 (link)} with a Zeiss AxioImager M2 microscope (Carl Zeiss, Inc) fitted with an AxioCam ICc1 digital camera. To analyse mosquito transmission, 30–50 Anopheles stephensi SD 500 mosquitoes were allowed to feed on anaesthetized, infected mice whose asexual parasitaemia had reached up to 15% and were carrying comparable numbers of gametocytes. To assess mid-gut infection, approximately 15 guts were dissected from mosquitoes on day 14 post feeding and oocysts were counted on Zeiss AxioImager M2 microscope. On day 21 post-feeding, another 20 mosquitoes were dissected and their guts and salivary glands crushed separately in a loosely fitting homogenizer to release sporozoites, which were then quantified using a haemocytometer or used for imaging. Mosquito bite back experiments were performed 21 days post-feeding using naive mice and blood smears were examined after 3–4 days.

Sporozoites were isolated from the salivary glands of mosquitoes infected with WTGFP and Δkinesin-20 parasites 21 dpi. Isolated sporozoites in RPMI 1640 containing 3% bovine serum albumin (Fisher Scientific) were pelleted (5 min, 5,000 rpm, 4°C) and used for motility assay. The assay using Matrigel was performed as described previously [19 (link),28 (link)]. A small volume (20 μl) of sporozoites, isolated as above for WTGFP and Δkinesin-20 parasites, were mixed with Matrigel (Corning, NY, USA). The mixture (6 μl) was transferred onto a microscope slide with a cover slip and sealed with nail polish. After identifying a field containing sporozoites, time-lapse videos (1 frame every 2 s for 100 cycles) were taken using the differential interference contrast settings with a 63× objective lens on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera and analysed with the AxioVision 4.8.2 software.
For ookinete motility, 24-h ookinete cultures were added to an equal volume of Matrigel on ice, mixed thoroughly, dropped onto a slide, covered with a cover slip, and sealed with nail polish. The Matrigel was then allowed to set at 20°C for 30 min. After identifying a field containing ookinetes, time-lapse videos were taken (1 frame every 5 s for 100 cycles).

Blood containing approximately 50,000 parasites of the kinesin knockout/knockdown lines was injected intraperitoneally (IP) into mice to initiate infection. Asexual stages and gametocyte production were monitored by microscopy on Giemsa-stained thin smears. Four to 5 dpi, exflagellation and ookinete conversion were examined as described previously [49 (link)] with a Zeiss AxioImager M2 microscope (Carl Zeiss) fitted with an AxioCam ICc1 digital camera. To analyse mosquito transmission, 30 to 50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetized, infected mice whose asexual parasitaemia had reached 15% and were carrying comparable numbers of gametocytes as determined on Giemsa-stained blood films. To assess midgut infection, approximately 15 guts were dissected from mosquitoes on day 7 and 14 post-feeding and oocysts were counted using a 63× oil immersion objective. On day 21 post-feeding, another 20 mosquitoes were dissected, and their guts and salivary glands crushed separately in a loosely fitting homogeniser to release sporozoites, which were then quantified using a haemocytometer or used for imaging. Mosquito bite back experiments were performed 21 days post-feeding using naive mice, and blood smears were examined after 3 to 4 days.

Phenotypic analysis was performed at different stages of the parasite life cycle as previously described (Roques et al., 2015). Briefly, asexual blood stages and gametocytes were analysed using infected blood smears. Gametocyte activation, zygote formation, and ookinete conversion rates were analysed using invitro cultures. For mosquito transmission, triplicate sets of 20–60 Anopheles stephensi were used. Briefly, exflagellation was examined on Days 4 to 5 post infection. Gametocyte‐infected blood was obtained from the tail with a heparinised pipette tip and mixed immediately with 40 μl of ookinete culture medium (RPMI1640 containing 25 mM HEPES, 20% fetal bovine serum, 10 mM sodium bicarbonate, and 50 μM xanthurenic acid at pH 7.6). Microgametogenesis was monitored at two different points during mitotic division (8 and 15 min post activation [mpa]). Gametocytes were purified and activated in ookinete medium then fixed and processed for immunofluorescence assay with antibodies to a range of different markers. Parasites were visualised on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera (Carl Zeiss, Inc).

Blood containing approximately 50,000 parasites of the CRK5-KO line was injected intraperitoneally into mice to initiate infections. Four to five days post infection, exflagellation and ookinete conversion were examined as described previously (Guttery et al., 2012 (link)) with a Zeiss AxioImager M2 microscope (Carl Zeiss, Inc) fitted with an AxioCam ICc1 digital camera. When indicated, gametocytes were pre-treated 5 min in SA supplemented with 1 µM BKI-1294 (Ojo et al., 2014 (link)) or 60 min in SA supplemented with 1 µM MG132. To analyse mosquito transmission, 30 to 50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetised, infected mice whose asexual parasitaemia had reached 15% and were carrying comparable numbers of gametocytes as determined on Giemsa-stained blood films. To assess mid-gut infection, approximately 15 guts were dissected from mosquitoes on day 7, 14, and 21 post-feeding and oocysts were counted using the Zeiss AxioImager M2 microscope and a 63x oil immersion objective. Mosquito bite-backs were performed 21 days post-feeding using naive mice and blood smears were examined after 3–4 days.

Neuro2A cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). Eagle’s minimal essential medium (EMEM) supplemented with 10 % fetal bovine serum (FBS), 2 mM l-glutamine and 50 units/ml of penicillin/streptomycin (complete EMEM) was used to maintain Neuro2A cells and stable transfected clones. Neuro2A cells were differentiated as previously described by Zeng and Zhou (2008 (link)). Briefly, cells were plated at a low density (100 cells/mm²) in 6-well plates in complete EMEM which was changed to the differentiation medium the next day (EMEM with 2 % FBS, 2 mM l-glutamine and 20 µM retinoic acid). Medium was replaced every day for 4 days. Micrographs showing cells at 4-day differentiation were collected by a Zeiss Axiovert 40 CFL microscope with a 10× objective (Carl Zeiss Ltd., Cambridge, UK). Images were taken using an AxioCam ICc 1 digital camera (Carl Zeiss Ltd.). Neurite outgrowth was assessed by taking four micrographs by random selection from all experimental conditions (in each case, at least 220 cells were sampled per experiment), and experiments were repeated three times. All cells possessing at least one neurite with a length at least twice the cell body were considered differentiated. Results were expressed as a percentage of differentiated/total cell number.

To initiate infections, blood containing 5×10⁶ parasites was injected i.p. into mice. Parasitemia and gametocytemia were determined on Giemsa stained blood films.
Once asexual parasitemia had reached ∼7% and numbers of gametocytes were comparable 30–50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetised infected mice.
Approximately 20 guts were dissected from mosquitoes on day 14 post feeding and mounted under Vaseline-rimmed cover slips after staining with Hoechst 33342 for 10–15 min to assess mid-gut infection. Oocysts were counted on an AxioCam ICc1 digital camera fitted to a Zeiss AxioImager M2 microscope using a 63x oil immersion objective.
On day 21 post-feeding another 20 mosquitoes were dissected and their guts and salivary glands crushed separately in a loosely fitting homogenizer to release sporozoites, which were then quantified using a haemocytometer.
Mosquito bite back experiments were performed on day 21 post feeding using naïve mice and blood smears were examined for the next 14 days to determine infection.