Hemolymph was collected from the dorsal side of the abdomen using pulled glass capillaries (Sutter Instrument Corp, Novato, CA, USA) following the procedure reported by Arafah et al. [29 (link)]. After collection, the hemolymph was immediately transferred to a chilled LoBind Protein microtube (Eppendorf, Germany) and pre-coated with phenylthiourea (PTU) and phenylmethylsulphonyl fluoride (PMSF) to prevent melanization and proteolysis, respectively. The hemolymph samples were frozen and stored at −20 °C until use (Figure 10 , step 3, hemolymph collection).
Lobind protein microtube
Manufactured by Eppendorf
Sourced in Germany
The LoBind Protein microtube is a laboratory equipment designed to minimize sample loss during storage and handling of sensitive biological samples, particularly proteins. The product features a specialized surface treatment that reduces the adsorption of proteins to the tube walls, helping to preserve sample integrity.
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2 protocols using lobind protein microtube
1
Hemolymph Collection for Proteomic Analysis
2
Honeybee Hemolymph Collection for Nosema Analysis
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Four days and thirteen days after Nosema spore inoculation, hemolymph from three (at 4 d.p.i.) and five honeybees (at 13 d.p.i.) per cage were collected (a total of 99 haemolymph samples and bled bees per condition at the end of the experiment; Figure 1).
For this part, no individual was collected at 7 d.p.i. to ensure sufficient number for the end of the experiment (13 d.p.i.). Honeybees were individually bled by piercing the abdomen dorsally between the second and third tergite using pulled glass capillaries (Sutter Instrument Corp, Novato, California). The hemolymph (~5 µL) was transferred into a chilled LoBind Protein microtube (Eppendorf, Germany) containing dried solutions of PTU at 20 µM and PMSF at 5 mM to minimize melanization and proteolysis, respectively. The hemolymph samples and the corresponding bled bodies were kept at -20ºC until further use. Samples with contamination from the gut or crop content were removed from the analyses.
For this part, no individual was collected at 7 d.p.i. to ensure sufficient number for the end of the experiment (13 d.p.i.). Honeybees were individually bled by piercing the abdomen dorsally between the second and third tergite using pulled glass capillaries (Sutter Instrument Corp, Novato, California). The hemolymph (~5 µL) was transferred into a chilled LoBind Protein microtube (Eppendorf, Germany) containing dried solutions of PTU at 20 µM and PMSF at 5 mM to minimize melanization and proteolysis, respectively. The hemolymph samples and the corresponding bled bodies were kept at -20ºC until further use. Samples with contamination from the gut or crop content were removed from the analyses.
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