Computational protocol: Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes

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[…] High-resolution microscopic imaging was used to confirm phagocytosis and incorporation of latex beads and CM-Dil (compare ). For both treatments, the collected and re-suspended hemolymph was pipetted on poly-L-lysine coated slides. The slides were kept in an incubator at 32°C for 40min in order to let the hemocytes adhere to the slide. Subsequently, slides were fixed for 20min with 4% paraformaldehyde (PFA, Sigma-Aldrich, Saint Louis, Missouri, USA) dissolved in phosphate buffered saline (PBS, Sigma-Aldrich, Saint Louis, Missouri, USA). Cells were permeabilized for 5min in 0.2% Triton-X 100 in PBS and washed 3 times for 5min in PBS. Finally, samples were co-stained for 45min with the nuclear marker (4',6-diamidino-2-phenylindole, DAPI; 1:1,000 from 0.5mg/mL stock; Sigma-Aldrich, Saint Louis Missouri, USA), and a marker for the cellular matrix protein F-actin (Alexa Flour 488 Phalloidin; Molecular Probes, Eugene, Oregon, USA). After staining, slides were washed 3 times for 5 min before they were mounted in 30% glycerol/PBS, and were sealed with nail polish.Confocal micrographs were acquired on a Leica TCS SP5 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). High-resolution images were taken with a 40x immersion oil objective (numerical aperture 1.25). The simultaneous acquisition mode was used to minimize spatial shifts between color channels; the z-step size was kept at 0.5μm. Signal detection bandwidth differed slightly between latex beads (excitation = 561nm, detection = 580-630nm) or CM-Dil stain (excitation = 561nm, detection = 570-617nm). Settings for the remaining two channels were kept constant, independent of treatment, namely 405nm/430-480nm for excitation/detection of DAPI and 488nm/580-650nm for excitation/detection of Alexa Flour 488 Phalloidin. Image processing with the ImageJ software package [] included maximum intensity projection of image stacks, and Gaussian filtering (kernel size = 1) to remove high spatial frequency noise.Morphological information from imaging allowed us to classify hemocytes by using established classification systems. Based on detailed descriptions for several insects [, , –], we identified granulocytes, plasmatocytes and prohemocytes in our samples from honey bees. [...] The T-test was used to test different phagocytic rate between CM-Dil and latex bead injections. Phagocytic rates in tests with E.coli pre-injection and two controls were contrasted using one-way ANOVA. Phagocytic rates and Vg levels in different worker types were analyzed by a one-way ANOVA, and post hoc Fisher Least Significant Difference (LSD) test. Possible links between phagocytosis and mitosis, as well as vitellogenin and mitosis were tested with Pearson’s correlation analysis. All analyses were performed with the Statistica 13.2 software package (Round Rock, TX, USA). […]

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