PMC:3625153 / 29152-30494 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3625153","sourcedb":"PMC","sourceid":"3625153","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3625153","text":"Haemocyte preparation and in vitro experiments\nHemolymph was extracted from the posterior adductor muscle of a number of individuals using a sterile 1-mL syringe then pooled to obtain the total volume required for each experiment. Hemolymph was then plated in 12-well plates (1 mL/well), and haemocytes were allowed settled for 1 h at 16°C in the dark. Cell attachment to the bottom of the well was checked microscopically. The medium was then removed, and cells were washed twice with 35-psu sterile artificial seawater (ASW). Control cells were incubated with 1 mL ASW, whereas 1 mL ASW containing the tested chemicals at the selected concentrations was added to the experimental wells. FSK and H89 were added to ASW from concentrated stock solutions prepared in dimethylsulphoxide (DMSO). In all cases, DMSO final concentration was 0.01% v/v and it did not significantly affect the biological endpoints analysed (data not shown). Experimental conditions (time of incubation and agonist/modulator concentrations) were assessed in preliminary trials to ensure significant evaluations of mRNA expressions along with cell signaling mediators. All incubations were carried out at 16°C in the dark. Each treatment consisted of 3 independent experiments, and each experimental trial consisted of 3 replicates for each biological endpoint (N = 3).","divisions":[{"label":"Title","span":{"begin":0,"end":46}}],"tracks":[]}