4 cells/microaggregate; Fluorescence microscopy: 105.6 nuclei/microaggregate ±8.8 nuclei/microaggregate; p>0.05; n=119). A minimum of four fields per slide area were counted per replicate (n≥4 replicates). To ascertain the contributions of CTX and its moieties to hemocyte adhesion, several protocols were used. In protocol one, to test if CTX caused hemocyte–substratum detachment, cells previously incubated with PBS (50 μl) were rinsed twice with PBS (2 ml), incubated with treatment solution (100 μl, 30 min at 37 °C, ∼95% RH), subsequently rinsed with PBS, fixed in glutaraldehyde
vapor, rinsed again with PBS, and mounted in 30% glycerol (v/v PBS). The total individually attached cells and their type (cells/mm2) were determined under by phase contrast microscopy. Protocol two, used to explain the initial hemocyte adhesion results
at 1.2 nM CTX, involved testing for XL184 supplier hemocyte lysis or reduced adhesion. The number of non-attached hemocytes after CTX treatment was determined on an unwashed hemocyte preformed monolayer. Without rinsing the slides, non-attached hemocytes were resuspended with gentle pipetting, collected (∼100 μl), added to areas on new slides, and centrifuged on a tissue culture plate rotor (500g, 5 min) to immobilize them on the slide. Hemocytes were fixed in 4% formaldehyde (in PBS) and mounted in 30% glycerol–PBS
buffer (v/v PBS) and the total number of cells/mm2, individual and aggregated, determined as previously described. For this protocol, the non-attached hemocytes (cells/mm2) results from the Bcl-2 inhibitor control buffer (PBS) were subtracted from values obtained from treatment values (1.2 nM CTX) because there may exist sub-populations of G. mellonella hemocytes, as alluded to for M. sexta hemocytes , which do not adhere to glass. Lepirudin Protocol three determined the effects of selected concentrations of RGDS and RGES (2.5 and 5.0 mM) on CTX (1.2 and 120 nM)-induced in vitro hemocyte microaggregation. Hemocytes were concomitantly treated with CTX and RGDS. Following treatment, hemocytes were immediately fixed in glutaraldehyde vapor for 30 min. Because soluble RGDS can inhibit hemocyte attachment and spreading onto polystyrene surfaces , hemocytes on slides were not rinsed and to avoid dispersal of individual cells and microaggregates due to direct coverslip contact, a coverslip supported above the reaction site was used and the microaggregate frequency determined by phase contrast microscopy. Since hemocytes per microaggregate using this method could not be accurately determined the total aggregated area (cumulative area of all microaggregates in a field) was measured by phase contrast microscopy and quantified with software (Adobe Photoshop CS3).