Micro Niche Finder 4.6.4 – Activation Included Serial Key [UPD]
Micro Niche Finder 4.6.4 – Activation Included Serial Key
methods: t. gondii tachyzoites (rh strain) were maintained by serial passage in hff monolayers at 37c in a humid 5% co2 atmosphere. three days after peal, a billion parasites were washed and cultured in dmem hg for 2h at 28c. supernatants free from parasites were filtered in a 0.45 m membrane, and evs isolation was performed using total exosome isolation kit. the yields were assessed by nanosight lm10. dimensional characterisation was performed using nanosight lm10. tem was carried out in jeol jem-1400 plus em. the protein levels were assessed by the qubit fluorometric quantification. three ev sample preparations, independently obtained from t. gondii, hff-infected, and non-infected cells were separated by sds-page. the gel lanes were excised, sliced and digested with trypsin. five micrograms of protein were analysed in triplicate by lc-ms/ms in a thermo scientific easy-nlc 1000 system coupled to a ltq orbitrap xl etd. peaklist picking, protein identification, were done using the maxquant version 1.5.0.25 and pattern lab platform.
results: with the present experimental conditions, a consistent ev yield of 1×106 particles/ml was obtained. it is important to note that our method provides a compromise between ev yield, quality and cost. nanosight technology allows a quick dimensional characterization by analysing four ev parameters such as size, shape, concentration and particle number. both non-infected and infected samples appeared well-described as, for example, the particles from non-infected cells showed spherical and elongated shape while the particles from infected-cell supernatants appeared mostly round, characteristic of membrane vesicles. tem pictures were consistent with nanosight analysis. unfortunately, since some proteins such as calnexin and gapdh were below the limits of detection of the current method, we were unable to compare the expression of the two proteins in the two experimental conditions.
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