Computational protocol: Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases

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Protocol publication

[…] Sand fly females were fed on anesthetized mice and dissected at different times post-blood meal (pbm). Dissection of midguts was semi-sterile and performed on ice, the preparation of one sample took maximum 30 min and the samples were immediately placed in a freezer (-80 °C). In experiments, midguts were homogenised while defrosting, centrifuged (12,000× g) and the supernatant was incubated with parasites.In the first series of experiments, various L. donovani (GFP) or L. major (dsRed) stages were incubated with P. argentipes, P. orientalis, P. papatasi or S. schwetzi midgut homogenates (dissected 24 h pbm) for 2 h in a microwell plate (1000 Leishmania/midgut). In the second series of the experiments, L. donovani (GFP) promastigotes (24 h after the release from macrophages) were incubated with P. argentipes, P. orientalis, P. papatasi or S. schwetzi midgut homogenates (dissected 6, 24, 32, 48 and 72 h pbm) for 2 h in a microwell plate (1000 Leishmania/midgut). In addition, promastigotes 24 h post-transition were incubated with proteinase K (6.7 mg/ml, 2 U/mg protein; Roche, Mannheim, Germany), rabbit blood, red cells of rabbit blood, human haemoglobin (220 mg/ml; Sigma-Aldrich), rabbit blood + proteinase K, red cells + proteinase K and haemoglobin + proteinase K. Leishmania parasites incubated for 2 h with saline instead of midgut homogenates were used as a negative control and parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 (Sigma-Aldrich) were used as a positive control.After the incubation, parasites with midgut homogenate were transferred to saline solution, dead cell were marked with DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride, 0.005 mg/ml; Thermo Fisher Scientific) and analysed by flow cytometry. Flow cytometry measurements were performed using flow cytometer CytoFLEX S (Beckman Coulter, Inc., Brea, California, USA) equipped with 4 lasers (405 nm, 488 nm, 561 nm and 638 nm) and 13 fluorescence detectors. GFP was excited using 488 nm laser and its fluorescence emission was detected using 525/40 filter, dsRed was excited using 561 nm laser and its fluorescence emission was detected using 585/42 bandpass filter, DAPI was excited by 405 nm laser and detected using 450/50 filter. Analysis of cytometry data was performed using CytExpert software (Beckman Coulter).The experiments were conducted in duplets and repeated twice. Statistical evaluations were performed by the ANOVA and post-hoc Tukey HSD test using SPSS Statistics 23.0 software. […]

Pipeline specifications

Software tools CytExpert, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Leishmania, Drosophila melanogaster, Leishmania donovani, Leishmania major, Toxoplasma gondii, Plectorhinchus orientalis, Phlebotomus papatasi, Oryctolagus cuniculus
Diseases Infection, Neoplasms